The influence of anoxia or oxygenation on the induction of chromosome aberrations in human lymphocytes by 15-MeV neutrons

Dicentric and total aberration yields induced in human lymphocytes by 15-MeV neutrons under conditions of oxygenation and of anoxia have been fitted to a dose-response curve using the function Y = D + βD². An oxygen-enhancement ratio (OER) ranging from 3.7 at low yields to 1.6 at high yields was calculated from the co-efficients of the dicentric yield curves and evidence is presented which suggests that oxygen does not act as a dose-modifying agent in this system. High dose RBE values of 1.2 and 2.1 with respect to 250 kVp X-rays for oxygenated and anoxic conditions were also obtained. Coefficients for total aberration yield gave similar values to dicentrics for both OER and RBE     

Temperature and the formation of radiation-induced chromosome aberrations. I. The effect of irradiation temperature

Irradiation temperature, changed from 37 degrees C to 4 degrees C, acts as a dose-modifying factor with regard to the dose-yield relationship for dicentric chromosome aberrations in human lymphocytes irradiated with 150 kV X-rays. The temperature dependence of the aberration yield observed at constant dose is S-shaped, with a sharp rise near 15 degrees C from a lower plateau below 12 degrees C to a higher plateau beyond 17 degrees C. The aberration yield is determined by the irradiation temperature, irrespective of fast temperature changes from 4 degrees C to 37 degrees C or from 37 degrees C to 4 degrees C, applied at various delay times before and after irradiation. It is concluded that irradiation temperature influences the formation of chromatin lesions rather than their interaction.     

Chromosome aberration yields induced in human lymphocytes by 15 MeV electrons given at a conventional dose-rate and in microsecond pulses.

Yields of unstable chromosome aberration were analysed in human lymphocytes after in vitro exposure to 15 MeV electrons. Two dose-effect curves were prepared. In one, doses of 44 to 742 rad were given at 100 rad/min, and in the other doses of 53 to 764 rad were each delivered in single microsecond pulses. No significant difference could be found between the two sets of data when analysed in terms of the quadratic model of aberration production. Good agreement was observed with other dose-response studies in this laboratory, in which human lymphocytes were exposed to 250 kVp X-rays and 60CO gamma-rays at conventional rates of 100 and 50 rad/min, respectively. Comparison with the results of a low-LET dose-rate experiment shows that the yield of dicentric aberrations remains constant overa wide range, i.e. 25 to 6X 10(9), 100 to 1-5 X 10(10), and 150 to 3 X 10(10) rad/min, respectively, for doses of 100, 250 and 500 rad. Radiochemical consumption of oxygen occuring in the lymphocytes during the single microsecond exposures may amount to less than 5 per cent of the total oxygen present in the blood samples, immediately before irradiation. The data also indicate that the ultra-high dose-rates currently available are insufficient to overcome the therapeutic problem of hypoxic radioresistant tumour cells.     

Oxygen enhancement ratio as a function of dose and cell cycle phase for radiation-resistant and sensitive CHO cells

There is still controversy over whether the oxygen enhancement ratio (OER) varies as a function of dose and cell cycle phase. In the present study, the OER has been measured as a function of survival level and cell cycle phase using volume flow cell sorting. This method allows both the separation of cells in different stages of the cycle from an asynchronously growing population, and the precise plating of cells for accurate measurements at high survival levels. We have developed a cell suspension gassing and sampling system which maintained an oxygen tension less than 20 ppm throughout a series of sequential radiation doses. For both radiation-resistant cells (CHO-K1) and a radiation-sensitive clone (CHO-xrs6), we could separate relatively pure populations of G1-phase, G1/S-boundary, S-, and G2-phase cells. Each cell line showed a typical age response, with cells at the G1/S-phase boundary being 4 (CHO-K1) to 12 (CHO-xrs6) times more sensitive than cells in the late S phase. For both cell lines, G1-phase cells had an OER of 2.3-2.4, compared to an OER of 2.8-2.9 for S-phase and 2.6-2.7 for G2-phase cells. None of these age fractions showed a dependence of OER on survival level. Asynchronously growing cells or cells at the G1/S-phase boundary had an OER similar to that of G1-phase cells at high survival levels, but the OER increased with decreasing survival level to a value near that of S-phase cells. These results suggest that the decrease in OER at high survival levels for asynchronous cells may be due to differences in the OERs of the inherent cell age subpopulations. For cells in one cell cycle stage, oxygen appears to have a purely dose-modifying effect.     

Chromosome aberration yields in human lymphocytes induced by fractionated doses of x-radiation.

Unstimulated (G0) human peripheral blood lymphocytes were exposed at 37 degrees C to doses of 200 or 500 rad of X-rays delivered in two equal fractions. The dose fractions were separated by intervals of up to 7 h in the 200 rad study and up to 48 h for 500 rad. In both studies the mean levels of dicentrics and total unstable aberrations began to decline when fractions were delivered with intervals of greater than 2 h. With 200 rad the yield had decreased to an additive baseline (i.e. equal to only twice the yield of a single 100-rad fraction) by an interval of 4 h. Following 500 rad the yield declined until 8 h and then remained 20% above the additive baseline even when 48 h separated the fractions. Possible explanations for this discrepancy are discussed. In a second experiment PHA stimulated lymphocyte cultures were exposed to 2 doses of 125 rad of X-rays up to 7 h apart in an attempt to demonstrate the late peak in aberration yields originally reported by Lane [5]. Control cultures received unsplit doses of 250 rad at the time of the corresponding second 125-rad fraction. No evidence of a late peak in dicentric yield was observed. The yield remained approximately the same irrespective of the time interval between fractions but these split dose yields were significantly different from the accompanying unsplit controls.     

The effect of X-irradiation on cell cycle progression and chromatid aberrations in stimulated human lymphocytes using cohort analysis studies.

Stage sensitivity for the production of chromatid-type aberrations and mitotic delay has been investigated in a stimulated human lymphocyte population, following an absorbed dose of 1.5 Gy 250 kVp X-rays. BrdU replication banding was used to obtain a fine analysis of the cell cycle and to permit cohort analysis. Fluctuations in yield with sample time were found for all aberration categories, but these could not be related simply to either the developmental stage of the cells at time of exposure, or to the time-to-run to metaphase. In general G2 and late S cells had higher aberration yields than early S and pre S cell populations. Mitotic delay and perturbation at this dose extends to all sub-phases of S and is as great, if not greater, in the earliest S cells as it is in G2.     

Chromosome aberration analysis and the influence of mitotic delay after simulated partial-body exposure with high doses of sparsely and densely ionising radiation

The influence of high doses of sparsely and densely ionising radiation on the yield of aberrant human peripheral lymphocytes in simulated partial-body exposures was studied by investigating radiation-induced chromosome aberration frequencies, namely dicentric and centric ring chromosomes. Peripheral blood samples from two volunteers were irradiated with high doses of 200 kV X-rays or neutrons with a mean energy of <E _n>=2.1 MeV and partial-body exposure was simulated by mixing irradiated and non-irradiated blood from the same two donors in proportions of 25, 50, and 75%. Lymphocytes were cultured and first-division metaphase cells were collected after culture times of 48, 56, and 72 h. A significant underrepresentation of dicentric and centric ring chromosomes was observed at the three highest doses of X-rays between the different culture times for nearly all proportions. After neutron irradiation, some significant differences were observed at all doses and all culture times, without however, revealing any systematic pattern. The distribution of dicentric and ring chromosomes showed overdispersion for both radiation types. After simulated partial-body exposures with 200 kV X-rays and <E _n>=2.1 MeV neutrons, strong mitotic delays could be observed, which depended on both the irradiated volume and the applied dose: the smaller the irradiated volume and the higher the dose, the higher was the selective advantage of non-irradiated cells. For the purpose of biological dosimetry after partial body exposure, an extension of the lymphocyte culture time is suggested at least for doses ≥3.0 Gy of 200 kV X-rays and ≥0.5 Gy of <E _n>=2.1 MeV neutrons in order to prevent a systematic underestimation of cytogenetic damage.     

A model for interphase chromosomes and evaluation of radiation-induced aberrations

We have developed a theoretical model for evaluating radiation-induced chromosomal exchanges by explicitly taking into account interphase (G(0)/G(1)) chromosome structure, nuclear organization of chromosomes, the production of double-strand breaks (DSBs), and the subsequent rejoinings in a faithful or unfaithful manner. Each of the 46 chromosomes for human lymphocytes (40 chromosomes for mouse lymphocytes) is modeled as a random polymer inside a spherical volume. The chromosome spheres are packed randomly inside a spherical nucleus with an allowed overlap controlled by a parameter Omega. The rejoining of DSBs is determined by a Monte Carlo procedure using a Gaussian proximity function with an interaction range parameter sigma. Values of Omega and sigma have been found which yield calculated results of interchromosomal aberration frequencies that agree with a wide range of experimental data. Our preferred solution is one with an interaction range of 0.5 microm coupled with a relatively small overlap parameter of 0.675 microm, which more or less confirms previous estimates. We have used our model with these parameter values and with resolution or detectability limits to calculate yields of translocations and dicentrics for human lymphocytes exposed to low-LET radiation that agree with experiments in the dose range 0.09 to 4 Gy. Five different experimental data sets have been compared with the theoretical results. Essentially all of the experimental data fall between theoretical curves corresponding to resolution limits of 1 Mbp and 20 Mbp, which may reflect the fact that different investigators use different limits for sensitivity or detectability. Translocation yields for mouse lymphocytes have also been calculated and are in good agreement with experimental data from 1 cGy to 10 cGy. There is also good agreement with recent data on complex aberrations. Our model is expected to be applicable to both low- and high-LET radiation, and we include a sample prediction of the yield of interchromosomal rejoining in the dose range 0.22 Gy to 2 Gy of 1000 MeV/nucleon iron particles. This dose range corresponds to average particle traversals per nucleus ranging from 1.0 to 9.12.     

The regulation of human exposure to mutagens amidst scientific controversy

Human peripherial lymphocytes were irradiated with different doses of 15.0-MeV neutrons. The frequency of different aberration types was determined and the dose-response relation was calculated. The data were fitted by least-squared regression analysis to different models. The dicetric, dicentric+centric ring, and different acentric data gave the best fit to the linear quadratic model. The RBE of 15.0-MeV neutrons versus 220 kV X-rays decreased significantly with increasing dose.     

Distribution pattern and dose-response-relationship of chromosome aberrations in human lymphocytes induced in vitro by 60Co gamma-rays and 110 kV X-rays.

Peripheral blood lymphocyte culture system was used to construct reference dose-response curves for 60Co gamma-rays and 110 kV X-ray-induced chromosome aberrations at 6 dose points ranging from 0.25 to 4.0 Gy. Qualitative and quantitative differences between these two types of radiation for the yield of induced aberrations and their distribution pattern were analysed. Experimental data of aberration yields were compared after fitting them to five different dose-response models. The yields of chromosome aberrations in particular dicentrics, gave a good fit to linear-quadratic besides linear and power models. In this model, single-track events predominated over double-track events for both the qualities of radiation used. The pattern of distribution was mainly Poisson for dicentrics but gave a conflicting result for acentrics which was in excess.     

Reduced oxygen enhancement ratio at low doses of ionizing radiation

A decreased oxygen enhancement ratio (OER) at lower radiation doses has been previously reported (B. Palcic, J. W. Brosing, and L. D. Skarsgard, Br. J. Cancer 46, 980-984 (1984]. The question remained whether or not this effect is due to a possible oxygen contamination at low doses, which was not the case at high doses. To ensure a sufficient degree of hypoxia prior to the start of irradiation, Chinese hamster cells (CHO) were made hypoxic by gas exchange combined with metabolic consumption of oxygen at 37 degrees C. At the same time oxygen levels in cell suspension were measured using a Clark electrode. It was found that under experimental conditions used in this laboratory for hypoxic irradiations, the oxygen levels before the start of irradiation are always below the levels which could give any significant enhancement to radiation inactivation by X rays. Full survival curves were determined in the dose range 0-30 Gy using the conventional survival assay and in the dose range 0-3 Gy using the low dose survival assay. The results confirmed the earlier finding that the OER decreases at low doses. It is therefore believed that the dose-dependent OER is a true radiobiological phenomenon and not an artifact of the experimental method used in the low dose survival assay.     

In vitro transmission of chromosomal aberrations through mitosis in human lymphocytes

Stable and unstable chromosome aberrations in human lymphocytes exposed to 2 and 4Gy of X-rays in G(0) were analyzed in M1 and M2 cells harvested at 72h to investigate how the scoring protocol influences the yields of aberrations transmitted through one mitosis. Metaphase chromosomes 2, 3, and 5 were painted using fluorescence in situ hybridization (FISH) whole chromosome probes, together with a pan-centromeric probe and stained by the harlequin-FISH method, to allow the cell cycle status of each cell to be determined as it was scored. A strict scoring criterion was adopted so that each metaphase had to contain 46 centromeres and each dicentric/centric ring had to have an acentric present. In addition to scoring the painted material, unstable aberrations in the whole genome were also recorded. The yield of complete dicentrics decreased by more than a factor of 2 in going from M1 to M2. The decrease was greater at the lower dose. Two-way translocations appeared stable, but one-way translocations decreased. This suggests that if translocation yields are to be used for biological dosimetry purposes, then the two-way type should be used.     

Chromosome aberrations induced in human lymphocytes by in vitro and in vivo X-rays

A dose-effect curve is presented obtained by analysis of dicentric chromosomes and centric ring chromosomes in lymphocyte metaphase spreads of three healthy volunteers after in vitro 100 kV X-ray-irradiation of peripheral blood samples. This calibration curve follows a linear quadratic equation, y=c+alpha D+beta D(2), with the coefficients: y=(0.0005+/-0.0001)+(0.0355+/-0.0066)D+(0.0701+/-0.0072)D(2). The model is based on 13.231 first-division metaphases analyzed after in vitro exposure to doses ranging from 0.1 to 2.0 Gy at a dose rate of 0.4 Gy min(-1). Significant overdispersion of the observed chromosomal aberrations was evident for dose points 1.0 and 2.0 Gy, respectively. The calibration curve was applied to derive equivalent whole body doses of three subjects after suspected extensive exposure to diagnostic X-rays.     

3-Aminobenzamide does not affect X-ray-induced cytogenetic damage in G0 human lymphocytes

The inhibition of poly(ADP-ribose) polymerase by 3-aminobenzamide (3AB) has been reported to have very different effects on X-ray-induced chromosome aberrations in G0 human lymphocytes. One group of investigators observed a 2-3-fold increase in the yield of rings, dicentrics and chromosome breaks after X-irradiation and 3AB treatment, whereas another group found that 3AB had no effect on X-ray-induced chromosome aberrations. To resolve this discrepancy, we repeated the experiments as described by both groups and found no effect of 3 mM or 5 mM 3AB on the frequency of chromosome aberrations induced by either 1 Gy or 2 Gy of X-rays. Furthermore, we found no effect of 3AB on X-ray-induced aberration yields in C-banded prematurely condensed chromosome preparations from unstimulated human lymphocytes. These results indicate that poly(ADP-ribose) polymerase is not involved in the repair of cytogenetic damage in G0 human lymphocytes.     

A New Model of Biodosimetry to Integrate Low and High Doses

Biological dosimetry, that is the estimation of the dose of an exposure to ionizing radiation by a biological parameter, is a very important tool in cases of radiation accidents. The score of dicentric chromosomes, considered to be the most accurate method for biological dosimetry, for low LET radiation and up to 5 Gy, fits very well to a linear-quadratic model of dose-effect curve assuming the Poisson distribution. The accuracy of this estimation raises difficulties for doses over 5 Gy, the highest dose of the majority of dose-effect curves used in biological dosimetry. At doses over 5 Gy most cells show difficulties in reaching mitosis and cannot be used to score dicentric chromosomes. In the present study with the treatment of lymphocyte cultures with caffeine and the standardization of the culture time, metaphases for doses up to 25 Gy have been analyzed. Here we present a new model for biological dosimetry, which includes a Gompertz-type function as the dose response, and also takes into account the underdispersion of aberration-among-cell distribution. The new model allows the estimation of doses of exposures to ionizing radiation of up to 25 Gy. Moreover, the model is more effective in estimating whole and partial body exposures than the classical method based on linear and linear-quadratic functions, suggesting their effectiveness and great potential to be used after high dose exposures of radiation.     

Algorithms for the evaluation of radiation induced chromosome aberration yields per cell from flow karyotypes

CHO-K1 cells were irradiated in G0/G1-phase with 150 kV X-rays. Single chromosomes isolated from metaphase cells and stained with DNA intercalating dye DAPI were analyzed in the ICP 22 with a modified flow chamber. In order to study dose-dependent changes in the flow karyotypes, they were split into peak- and background-portions by an iterative fit algorithm. As in a first approach, estimates of the frequencies of chromosome lesions were derived from an evaluation of the dose-dependent reduction in peak contents. The number of radiation-induced lesions per chromosome was found to be proportional to its length. As a second approach, the number of fluorescence events in the histogram background was corrected for non-chromosomal debris and evaluated interms of chromosome aberration frequency per cell, which was consistent with the yields of dicentric chromosomes and acentric fragments observed in microscopic investigations. As a third approach, lesion frequencies were calculated from the corrected background light sum in the karyotypes, utilizing a Monte Carlo model to simulate the effect of aberration formation on the flow histogram. The results indicate that the number of chromosome lesions observed by flow cytometry can be quantitatively related to the yield of structural chromosome aberrations detected by microscopic analysis. Dose-effect relations and split-dose kinetics are given as examples demonstrating the usefulness of this technique in radiobiology. Time saving compared to microscopic analysis was of the order of 90%.     

Interaction between X-ray and alpha-particle damage in V79 cells

V79 cells have been exposed to X-rays or 238Pu alpha-particles or to X-rays following priming alpha-particle doses of 0.5, 2 or 2.5 Gy. The survival curve for exposure to alpha-particles was exponential with a D0 of 0.89 Gy. Following exposure to priming alpha-particle doses the resulting X-ray survival curves had the same slope as the single dose X-ray curve, but a reduced shoulder. For alpha-particle priming doses of 0.5 and 2 Gy this reduction was the same as for the same X-ray doses. 2.5 Gy alpha-particles reduced the subsequent X-ray curve Dq to almost zero. alpha-particles do cause damage capable of interacting with X-ray damage.     

Interlaboratory comparison of the dicentric chromosome assay for radiation biodosimetry in mass casualty events

This interlaboratory comparison validates the dicentric chromosome assay for assessing radiation dose in mass casualty accidents and identifies the advantages and limitations of an international biodosimetry network. The assay's validity and accuracy were determined among five laboratories following the International Organization for Standardization guidelines. Blood samples irradiated at the Armed Forces Radiobiology Research Institute were shipped to all laboratories, which constructed individual radiation calibration curves and assessed the dose to dose-blinded samples. Each laboratory constructed a dose-effect calibration curve for the yield of dicentrics for (60)Co gamma rays in the 0 to 5-Gy range, using the maximum likelihood linear-quadratic model, Y = c + alphaD + betaD(2). For all laboratories, the estimated coefficients of the fitted curves were within the 99.7% confidence intervals (CIs), but the observed dicentric yields differed. When each laboratory assessed radiation doses to four dose-blinded blood samples by comparing the observed dicentric yield with the laboratory's own calibration curve, the estimates were accurate in all laboratories at all doses. For all laboratories, actual doses were within the 99.75% CI for the assessed dose. Across the dose range, the error in the estimated doses, compared to the physical doses, ranged from 15% underestimation to 15% overestimation.     

Remarks on oxygen effects

Oxygen concentration will influence the alpha- and beta-effect (two-component theory of radiation effects) independently. The beta-effect is reduced by a dose-modifying factor gamma. Oxygen, in competition with enzyme repair actions fortifies a part of the beta-lesions and this model leads to a simple equation for the factor gamma. The alpha-effect is also enhanced by oxygen. Measurements of OER indicate that the reduction of the effect from aerobic to anoxic condition might be about 70%. The functional dependence of oxygen concentration has not been investigated. For small oxygen concentrations of 0.15 to 0.5 muM/l and doses below 1000 R Révész and Littbrand have found that oxygen can protect the irradiated cells and thus increase survivals with about 10%. This is explained as a scavenger action where radical hydrogen atoms are oxidized and hydrated electrons captured by oxygen moelcules. When the oxygen concentration is increased, or with higher doses, the usual sensitizing effect of oxygen exceeds the protection effect. The influence of oxygen on alpha-effects are mainly connected with indirect radiation effects and thus depend on temperature and milieu.     

Rapid dicentric assay of human blood lymphocytes after exposure to low doses of ionizing radiation

The probability of losses of different chromosome aberrations during the dicentric chromosome assay of metaphase cells with incomplete sets of chromosome centromeres was estimated using a mathematical model for low doses of ionizing radiation. A dicentric assay of human blood lymphocytes without determination of the total amount of chromosome centromeres in cells without chromosome aberrations (rapid dicentric assay) has been proposed. The rapid dicentric analysis allows to register chromosome aberrations in full compliance with the conventional classification. The experimental data have shown no statistically significant difference between the frequencies of dicentric chromosomes detected by rapid and classical dicentric chromosome assays of human lymphocytes exposed to 0.5 Gy of 60Co gamma-rays. The rate of the rapid dicentric assay was almost twice as high as that of the classical dicentric assay.