Objective
Cytogenetic dosimetry following ionising radiation usually involves the analysis of metaphase chromosomes from mitogen stimulated peripheral blood lymphocytes. This project aimed at the development of a new cytogenetic approach for biological dosimetry which would allow the early assessment of radiation injury and the establishment of absorbed dose estimates in accidental overexposures. This new approach to biological dosimetry is based on the analysis of radiation induced chromosomal aberrations scored in C-banded peripheral blood lymphocyte prematurely condensed chromosomes (PCC).
The cytogenetic approach used in this project, which allows the direct observation of chromosome damage without having lymphocytes proceed to mitosis, can be very useful for biological dosimetry purposes. Ring and dicentric analysis in PCCs provides not only confirmatory evidence of an exposure, especially when blood samples are not available soon after irradiation, but also quantitative estimates in terms of equivalent whole body doses.
The identification of the sequence of events that lead from deoxyribonucleic acid (DNA) damage to chromosome damage and cell death is of particular importance for the elucidation of the action of ionising radiation in living cells. Experiments were carried out to study the ability of compounds acting via inhibition of DNA polymerases such as 9-B-D-arabinofuranosyladenine (ara A), 1-B-D-arabinofuranosyladenine (ara C) and aphidicolin, to inhibit repair of radiation induced damage at the DNA level, chromosomal level and cellular level in plateau phase Chinese hamster ovary (CHO) cells. For the experiments at the chromosomal level, the premature chromosome condensation (PCC) technique was used. Repair of total DNA breaks was measured by the unwinding technique and repair of DNA double strand breaks by the neutral filter elution technique.
The results suggest that repair and ara A mediated fixation of periodic lattice distortion (PLD) have their counterparts at the chromosome level as indicated by the similar repair kinetics an inhibition/fixation characteristics obtained for PLD and chromosome damage. Since ara A is expected to inhibit the polymerisation steps necessary for the completion of DNA repair, it is suggested that DNA polymerisation is required for chromosome repair. Among lesions induced by radiation in the DNA, double strand breaks are the lesions more likely leading to chromosome damage. Furthermore, the results obtained demonstrate that the efficacy of a polymerase inhibitor to inhibit DNA and chromosome repair does not always coincide with its ability to fix radiation induced PLD, or with its ability to inhibit DNA replication. This finding indicated that partly different biochemical pathways may underlie PLD fixation, DNA repair inhibition and DNA replication inhibition.
Incorporation of bromodeoxyuridine (BrdU) into deoxyribonucleic acid (DNA), in the place of thymidine, sensitises exponentially growing and plateau phase Chinese hamster ovary (CHO) cells to a subsequent exposure to low linear energy transfer (LET) radiation. An increase in the amount of DNA and chromosome damage induced per unit radiation dose was observed with increasing incorporation of BrdU into DNA that was quantitatively similar to the increase observed in the survival curve slope. Although sensitisation was observed both in cells irradiated in the exponential as well as in cells irradiated in the plateau phase of growth, the degree of sensitisation was significantly larger in exponentially growing cells for the same degree of thymidine replacement by BrdU in the DNA. It is hypothesised that this result indicates the possible importance of chromatin structure at the time of irradiation and/or the importance of chromatin conformation changes after irradiation in the expression of radiation induced potentially lethal damage in BrdU containing cells. BrdU incorporation affected both the slope and the shoulder width of the survival curve, and increased the induction of DNA and chromosome damage per unit absorbed dose. The increase observed in the survival curve slope was quantitatively similar to the increase observed in damage induction at the DNA and chromosomal level, suggesting a cause effect relationship between these phenomena. Reduction in the shoulder width did not correlate with the increase in DNA and chromosome damage induction suggesting that different phenomena, probably related with enhanced fixation of radiation induced periodic lattice distortion (PLD) in BrdU containing cells, underlie its modulation.
Xrs-5 cells are known to be deficient in DNA double strand break (DSB) repair. Compared to similar results obtained in similar stages of growth with repair proficient CHO cells, a reduction was observed with xrs-5 cells in BrdU induced radiosensit isation for similar levels of incorporation. BrdU incorporation did not affect the induction of DNA DSBs as measured by the nonunwinding filter elution technique, a result that contradicts findings with CHO cells. At the chromosomal level, an increase was observed in the induction of breaks in the BrdU containing cells, as measured in noncycling G1 cells using the premature chromosome condensation (PCC) technique. This increase in chromosome breakage was comparable to that observed in similar experiments with CHO cells. These results indicate similarities in the effect of BrdU at the chromosomal level in irradiated CHO and xrs-5 cells, suggesting that DNA DSB repair proficiency is a prerequisite for halogenated pyrimidine induced radiosensitisation.
Hyperthermia:
In order to study the effect of hyperthermia on the induction and repair of radiation induced chromosome damage, experiments were designed to study the effects of heat (43 and 45.5C) on chromatin morphology and nuclear organisation, as visualised by premature chromosome condensation (PCC), in exponentially growing cells and plateau Chinese hamster ovary (CHO) cells. Experiments were also carried out with exponentially growing HeLa cells.
The results obtained indicate that exposure to heat drastically reduces the ability of interphase chromatin to condense and the ability of the nucleolar organisingregion to disintegrate under the influence of factors provided by mitotic cells when fused to interphase cells. The fraction of cells with nondisintegrated nucleoli increased with increasing exposure time at 45.5 C and reached a plateau at almost 100% after about 20 minutes. Exponentially growing and plateau phase cells showed similar response. Recovery from the effects of heat on chromatin condensation and disintegration of the nucleolar organising region depended upon the duration of the heat treatment. For exposures up to 15 minutes at 45.5 C, a gradual reduction in the fraction of cells with nondisintegrated nucleoli was observed when cells were allowed for repair at 37 C. However, only a very limited amount of repair was observed after 30 minutes exposure to 45.5 C. The repair time observed at the chromosome level were similar to those reported for the removal of excess protein accumulating in chromatin or the nuclear matrix, suggesting a causal relationship between the 2 phenomna. It is proposed that nuclear protein accumulation on chromatin or in the nuclear matrix reduces the accessibility of chromatin to enzymes responsible for the phosphorylation reactions necessary for chromatin condensation and disintegration of the nucleolus.
Effect of hyperthermia on the induction and repair of radiation induced chromosome damage:
The effect of preexposure to heat on the induction and repair of chromosome damage was measured in plateau phase CHO cells using the PCC technique. Plateau phase cultures were obtained by growing 1E5 cells in T25 tissue culture flasks for 4 days without refeeding. Cells were exposed to heat (45.5 C) for 8 or 15 minutes in fresh growth medium without serum, were irradiated, either immediately or at various times thereafter (up to 16 hours), and were returned to the incubator at 37 C. At various time intervals after irradiation (up to 24 hours) flasks were trypsinised and an aliquot containing 1E6 cells were mixed with an equal amount of cells selected at mitosis using nocodazole. The cell mixture was treated with polyethylene glycol (PEG) to effect fusion and PCC induction. Exposure to heat prevented chromatin from fully condensing and nucleoli from disintegrating. Therefore, measurements of residual chromosome fragments were carried out after reversion of these effects. Despite these inherent difficulties in measuring chromosome breaks in interphase cells after exposure to heat, the results obtained clearly indicated a significant reduction in the ability of heated cells to repair radiation induced chromosome damage. The experiments also indicated a larger induction by radiation of chromosome damage in heated cells.
Induction and repair of chromosome damage were studied in interphase xrs-5 cells by means of the premature chromosome condensation (PCC) technique. The results obtained were compared to those previously reported for Chinese hamster ovary (CHO) cells. Induction of chromosome damage per unit of absorbed radiation dose was in xrs-5 cells larger by a factor of 2.6 than in CHO cells. Changes in chromatin structure, associated with the radiation sensitive phenotype of xrs-5 cells, that increase the probability of conversion of a deoxyribonucleic acid (DNA) double strand break (DSB) to a chromosome break are invoked to explain this effect. Repair of chromosome breaks as measured in plateau phase G1 cells was deficient in xrs-5 cells, and the number of residual chromosome breaks was practically identical to the number of lethal lesions calculated from survival data. This observation suggests than nonrepaired chromosome breaks are likely to be manifestations of lethal events in the cell. The yield of ring chromosomes scored after a few hours of repair was higher by a factor of 3 in xrs-5 compared to CHO cells. This increase in ring formation suggests an increase in the probability of misrepair of chromosome damage that may stem either from the reduced ability of xrs-5 cells to repair DNA DSB or from the higher production of chromosome fragments observed per cell and per Gy.
THIS RESEARCH PROGRAMME ON RADIATION CYTOGENETICS AND BIOLOGICAL DOSIMETRY HAS TWO MAIN OBJECTIVES. THE FIRST IS THE DEVELOPMENT OF A SENSITIVE AND RELIABLE BIOLOGICAL DOSIMETER FOR THE EARLY ASSESSMENT OF READIATION INJURY AND THE ESTABLISHMENT OF ABSORBED DOSE ESTIMATES IN ACCIDENTAL OVEREXPOSURES. A CYTOGENETIC APPROACH TO BIOLOGICAL DOSIMETRY WILL BE USED WHICH IS BASED ON RADIATION-INDUCED CHROMOSOMAL ABERRATIONS SCORED IN C-BANDED PERIPHERAL BLOOD LYMPHOCYTE PREMATURELY CONDENSED CHROMOSOMES (PCCS).
THE SECOND OBJECTIVE IS TO EXAMINE WHETHER CELL-CYCLE-DEPENDENT FLUCTUATIONS IN THE FIXATION AND/OR REPAIR OF RADIATION-INDUCED CYTOGENETIC LESIONS ARE REFECTED BY FLUCTUATIONS IN THE EXPRESSION AND/OR REPAIR OF POTENTIALLY LETHAL DAMAGE (PLD).
PHASE I: BIOLOGICAL DOSIMETRY ASPECTS
A. DEVELOPMENT OF A C-BANDING PROCEDURE FOR THE ACCURATE ANALYSIS OF CHROMOSOME ABERRATIONS IN PCCS.
THE QUANTITATION OF AN EXPOSURE BY MEANS OF THE PCC TECHNIQUE HAS BEEN LIMITED SO FAR TO THE ANALYSIS OF CHROMOSOME FRAGMENTS. THE YIELD OF FRAGMENTS, HOWEVER, DECREASES WITH TIME DUE TO REPAIR PROCESSES AND FORMATION OF EXCHANGES, SUCH AS CENTRIC RINGS AND DICENTRICS, WHOSE ANALYSIS REQUIRES UNAMBIGUOUS VISUALIZATION OF THEIR CENTROMERIC REGIONS (C-BANDING).
B. CONSTRUCTION OF IN VITRO CALIBRATION CURVES.
CENTRIC RINGS AND DICENTRIC ANALYSIS, ESPECIALLY WHEN BLOOD SAMPLES ARE NOT AVAILABLE SOON AFTER IRRADIATION, WILL PROVIDE NOT ONLY CONFIRMATORY EVIDENCE OF AN EXPOSURE, BUT ALSO QUANTITATIVE ESTIMATES IN TERMS OF EQUIVALENT WHOLE-BODY DOSES, IF REPRESENTATIVE CALIBRATION CURVES FOR DIFFERENT KIND OF RADIATION ARE ESTABLISHED. IN THIS RESEARCH PROGRAMME, SUCH DOSE-RESPONSE CURVES FOR THE MOST COMMON RADIATION SOURCES, DOSE RATES AND QUALITIES USED, WILL BE CONSTRUCTED AND COMPARED WITH THOSE ESTABLISHED USING THE CONVENTIONAL METAPHASE CHROMOSOME ANALYSIS.
1) CALIBRATION CURVES FOR X- AND GAMMA- IRRADIATION.
SAMPLES OF WHOLE HUMAN BLOOD WILL BE IRRADIATED WITH VARYING X- AND GAMMA- DOSES UP TO 1 GY AND INCUBATED AT 37 CELSIUS DEGREES FOR 6 HOURS, THUS ISOLATED AND PREPARED FOR CELL FUSION WITH MITOTIC CHO CELLS AND PCC INDUCTION. C-BANDED CHROMOSOME PREPARATIONS WILL BE ANALYSED FOR CENTRIC RINGS AND DICENTRIC PCCS.
2) CALIBRATION CURVES FOR DIFFERENT DOSE RATE AND RADIATION QUALITIES.
VARYING DOSE RATE OF X- AND GAMMA- RAYS, NEUTRONS, AND HEAVY IONS WILL BE USED.
C. ESTABLISHMENT OF RBE VALUES FOR LOW LEVEL DOSES.
DOSE-RESPONSE CURVES WILL BE ALSO ESTABLISHED FOR ACENTRIC FRAGMENTS SCORED IMMEDIATELY AFTER IRRADIATION SO THAT, TAKING ADVANTAGE OF HIGH SENSITIVITY OF THE PSS TECHNIQUE, ESPECIALLY WHEN APPLIED BEFORE A LARGE AMOUNT OF NORMAL REPAIR TAKES PLACE, RBE VALUES FOR LOW LEVEL DOSES OF DIFFERENT TYPES OF RADIATION USED WILL BE OBTAINED.
D. COMPARISON OF IN VIVO AND IN VITRO DOSE-RESPONSE CURVES.
TO EXAMINE WHETHER THERE ARE DIFFERENCES BETWEEN IN VITRO AND IN VIVO DOSE-RESPONSE RELATIONSHIPS, SPAGUE-DAWLEY RATS WITH THEIR CORRESPONDING IN VITRO BLOOD SAMPLES WILL BE EXPOSED TO DIFFERENT DOSE RATES AND RADIATION QUALITIES.
PHASE II: MECHANISMS OF RADIATION ACTION.
EXPERIMENTAL EVIDENCE SUGGESTS A CAUSAL RELATIONSHIP BETWEEN DNA DAMAGE, CHROMOSOME ABERRATION FORMATION AND CELL LETHALITY. NEVERTHELESS, EXPLANATIONS FOR THE VARYING RADIOSENSITIVITY OF CELLS IN G1, S AND G2, AND FLUCTUATIONS IN PLD EXPRESSION HAVE NOT BEEN WELL ESTABLISHED.
A. RADIATION-INDUCED CYTOGENETIC DAMAGE IN RELATION TO CHANGES IN INTERPHASE CHROMOSE CONFORMATION
RECENT EXPERIMENTAL EVIDENCE PROVIDED BY US SHOWS THAT THE YIELD OF CHROMOSOME FRAGMENTS OBSERVED IN INTERPHASE CELLS AFTER EXPOSURE TO A GIVEN X-RAY DOSE DEPENDS ON CHROMOSOME CONFORMATION AT THE TIME OF IRRADIATION AS WELL AS ON HOW SOON AFTER IRRADIATION THIS CHROMOSOME CONFORMATION CHANGES.
SYNCHRONIZED MITOTIC CHO CELLS WILL BE X-IRRADIATED AND THEIR PROGRESS TOWARDS CELL DIVISION AND G1 PHASE WILL BE DELAYED FOR VARIOUS TIME PERIODS BY MEANS OF COLCEMID OR NOCODAZOLE. ANALYSES WILL BE CARRIED OUT ON T
Fields of science (EuroSciVoc)
CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques. See: The European Science Vocabulary.
CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques. See: The European Science Vocabulary.
- natural sciences biological sciences genetics DNA
- natural sciences biological sciences genetics chromosomes
- natural sciences biological sciences biochemistry biomolecules proteins enzymes
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