The mouse splenocyte assay, an in vivo/in vitro system for biological monitoring: studies with X-rays, fission neutrons and bleomycin.

A modified mouse splenocyte culture system was standardized after testing different mitogens (i.e., phytohemagglutinin (PHA), concanavalin A (Con A)). The mitotic index was determined for comparison between different mitogens. Following selection of appropriate mitogen (PHA 16, Flow), a series of experiments were conducted to evaluate the application of a cytokinesis-block for scoring micronuclei and assays for chromosomal aberrations produced by treatment in G0 and G2 for the purposes of biological dosimetry following in vivo and/or in vitro exposure to X-rays, fission neutrons and bleomycin. In the X-irradiation studies, the frequencies of micronuclei and chromosomal aberrations (i.e., dicentrics and rings) increased in a dose-dependent manner. These data could be fitted to a linear-quadratic model. No difference was observed between irradiation in vivo and in vitro, suggesting that measurement of dicentrics and micronuclei in vitro after X-irradiation can be used as an in vivo dosimeter. Following in vivo irradiation with 1 MeV fission neutrons and in vitro culturing of mouse splenocytes, linear dose-response curves were obtained for induction of micronuclei and chromosomal aberrations. The lethal effects of neutrons were shown to be significantly greater than for a similar dose of X-rays. The relative biological effectiveness (RBE) was 6-8 in a dose range of 0.25-3 Gy for radiation-induced asymmetrical exchanges (dicentrics and rings), and about 8 for micronuclei in a dose range of 0.25-2 Gy. Furthermore, the induction of chromosomal aberrations by bleomycin was investigated in mouse G0 splenocytes (in vitro) and compared with X-ray data. Following bleomycin treatment (2 h) a similar pattern of dose-response curve was obtained as with X-rays. In this context a bleomycin rad equivalent of 20 micrograms/ml = 0.50 Gy was estimated.     

The cytogenetic effects of low doses of accelerated charged particles in human blood lymphocytes in vitro

The aim of the investigation was the study of cytogenetic effects in human blood lymphocytes of low doses of ionizing radiation in vitro. The analysis of unstable chromosome aberrations in human lymphocytes after irradiation by the accelerated ions 12C with the energy 500 MeV/nucleon and LET 10.7 keV/microm was carried out. Blood samples were irradiated on Nuclotron of the High Energy Laboratory of the Joint Institute for Nuclear Research. The doses of irradiation were in the range from 0.05 up to 1.0 Gy. Was shown that the frequency of unstable chromosome aberrations depends from the dose of ionizing radiation and can be described by linear function. At the doses 0.25-0.50 Gy the dose-independent curve was obtained for dicentrics and centric rings. The frequencies of dicentrics and centric rings as markers of the radiation action were slightly different for different donors that could be explained by different radiosensitivity. Using the calibration curve obtained earlier for gamma-rays coefficients of relative biological efficiency of accelerated 12C with the energy 500 MeV/nucleon were defined: they varied from 1.0 at the doses (0.5-1.0 Gy) up to 3.2 at the lower doses (0.05-0.25 Gy).     

Genotoxic effects of radiotherapy and chemotherapy on the circulating lymphocytes of breast cancer patients. I. Chromosome aberrations induced in vivo

Unstable chromosome aberrations were scored in peripheral blood lymphocytes (PBL) serially collected from 21 breast cancer patients before and after radiotherapy (RT), chemotherapy (CT) and combined treatments. Local radiotherapy as treatment for mammary cancer induced unstable chromosome aberrations in peripheral blood lymphocytes. Only a fraction of these lymphocytes were exposed to irradiation during treatment and the chromosomal damage observed in PBL was equivalent to that induced by irradiation in vitro with 2 Gy at high dose rate, i.e., about 4% of the total dose delivered locally. Chemotherapy alone did not induce such anomalies. Apart from the observed interindividual variations in either the level or the fate of dicentrics with time, different features of chromosome damage were found when chemotherapy was given before or after local cobaltotherapy: secondary chemotherapy did not alter the frequency and the overdispersed distribution of dicentrics observed after first-line radiotherapy; in contrast, when CT was given before radiotherapy, a lower dicentric frequency was scored, the distribution of dicentrics was not always found to be overdispersed and there was a time-dependent decrease in dicentrics after in vivo exposure.     

Characteristic cytogenetic effects of small doses of ionizing radiation

A study was made of the frequency of chromosome aberrations in human lymphocyte culture after gamma-irradiation (60Co) with doses ranging from 0.05 to 1.0 Gy at dose--rates of 0,005, 0.05 and 0.5 Gy/min. The frequency of structural changes in chromosomes at low doses was higher than it was expected in the case of extrapolating the effect produced by high to low doses of radiation; within the dose range from 0.1 to 0.5 Gy a plateau was registered for aberrations of the exchange type (dicentrics and rings). The abnormal character of the dose dependence of the yield of chromosome aberrations persisted with all three dose - rates under study.     

Comparison of cytogenic response of human lymphocytes to in vivo and in vitro exposure to low doses of gamma-rays. Unstable chromosomal exchanges detected by FPG techniques

On peripheral lymphocytes of eight cancer patients undergone whole-body therapeutic irradiation (at daily dose of 10 cGy up to total dose of 50 cGy of 60Co gamma-rays) the dose-response of unstable chromosome exchanges (dicentrics and centric rings) was studied. This dose response fitted well linear function. The lower slope of dose-response curve was found for in vivo irradiated lymphocytes as compared to the dose response curve obtained for in vitro irradiated lymphocytes of the same patients. This finding seems to provide evidence that in case of protracted irradiation of individuals an absorbed dose could be underestimated if for biological dosimetry an in vitro dose response curve for unstable chromosome aberrations is used as referent one.     

The characterization and transmissibility of chromosome aberrations induced in peripheral blood lymphocytes by in vitro alpha-particle radiation

Peripheral blood lymphocytes were irradiated in vitro with (213)Bi alpha particles at doses of 0, 10, 20, 50, 100, 200 and 500 mGy. Chromosome analysis was performed on 47-h cultures using single-color fluorescence in situ hybridization (FISH) to paint chromosomes 1, 3 and 5. The whole genome was analyzed for unstable aberrations to derive aberration frequencies and determine cell stability. The dose response for dicentrics was 33.60 +/- 0.47 x 10(-2) per Gy. A more detailed analysis revealed that the majority of aberrations scored as dicentrics were part of complex/multiple aberrations, with the proportion of cells containing complexes increasing with dose. Cells containing aberrations involving painted chromosomes (FISH aberrations) were further classified according to cell stability and complexity. The majority of cells with FISH aberrations were unstable. The proportion of aberrant FISH cells with complex/multiple aberrations ranged from 56% at 10 mGy to 89% at 500 mGy. A linear dose response for genomic frequencies of translocations in stable cells fitted the data from 0 to 200 mGy with a dose response of 7.90 +/- 0.98 x 10(-2) per Gy, thus indicating that they are likely to be observed in peripheral blood lymphocytes from individuals with past or chronic exposure to high-LET radiation. Comparisons with the dose response for low-LET radiation suggest an RBE of 13.6 for dicentrics in all cells and 3.2 for translocations in stable cells. Since stochastic effects of radiation are attributable to genetic changes in viable cells, translocations in stable cells may be a better measure when considering the comparative risks of different qualities of radiation.     

The induction of chromosome aberrations during the course of radiation therapy for morbus Hodgkin

In a patient with Morbus Hodgkin, structural aberrations of the chromosome type in peripheral lymphocytes were analyzed during radiation therapy (accumulated target dose 44.6 Gy: 22 fractions of 1.8 Gy each and 2 fractions of 2.5 Gy each at the end of the therapy). The blood was sampled about 5 min after a fraction and/or 24, 48, or 72 h thereafter. The frequency of dicentric chromosomes:acentric fragments:centric ring chromosomes is 37:14:1 throughout the therapy. Independent of the time of blood sampling after a fraction, the distributions of dicentrics and acentrics are overdisperse and represent negative binomial distributions. The yields from these aberrations, as determined during the course of radiotherapy, are best fitted to a linear-quadratic function with a negative quadratic term. The two dose-effect curves (blood sampling about 5 min and 24 to 72 h after a fraction) of dicentrics and acentrics do not differ significantly. Up to an accumulated target dose of about 20 Gy the percentages of cells with chromosome aberrations increase to about 48 to 65% and, at this level, remain constant until the end of therapy.     

A method of estimating the dose to and magnitude of an irradiated area during partial radiation damage based on the results of a cytogenetic analysis of a culture of peripheral blood lymphocytes

The curves, obtained in the in vitro experiments, that show the dependence of the frequency of dicentrics (per a cell with dicentrics) upon dose and percentage of cells with dicentrics are proposed to be used in estimating a dose and body volume affected by partial irradiation (an extreme case of nonuniform exposure) by the analysis of chromosome aberrations in peripheral blood lymphocyte cultures.     

Comparison of cytogenetic response of human lymphocytes to in vivo and in vitro exposure to low-dose gamma rays. Translocations and dicentrics detected by the FISH technique

On peripheral lymphocytes of 5 cancer patients undergone wholebody therapeutic irradiation (at daily dose of 10 cGy up to total dose 50 cGy of 60Co gamma-rays) the dose response of unstable and stable chromosomal exchanges detected by FISH was compared with the dose response of the some aberrations in lymphocytes irradiated in vitro. The dose response fitted well to linear function. For dicentrics the lower slope of dose-response curve was found for in vivo irradiated lymphocytes as compared to the dose-response curve obtained for in vitro irradiated lymphocytes of the same patients. No difference between in vivo and in vitro irradiation of lymphocytes was found for translocations. The frequency of translocations increased faster with the dose than the frequency of dicentrics only in lymphocytes irradiated in vivo.     

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.     

The clastogenic effect of irradiated human plasma

Normal unirradiated human lymphocytes were cultured in medium containing 20 per cent homologous or autologous plasma collected from samples of blood exposed in vitro to various doses of X-irradiation. Metaphases were stained by the BrdU/FPG method. The yields of chromatid-type aberrations in cells at first mitosis (M1 cells) were similar for cultures containing plasma irradiated at 0, 0.05 or 0.25 Gy but were significantly increased at 0.5, 5.0 and 10.0 Gy. The response was dose dependent but the data were insufficient to propose a particular model of dose response. The absence of chromosome-type aberrations confirmed the suggestion that earlier workers' observations of dicentrics and rings were artefacts of long culture times. The level of chromosomal damage was unaffected by omitting folic acid from the medium. Irradiated plasma did not alter the frequency of sister chromatid exchange observed in M2 cells. The ratios of M1, M2 and M3 cells were markedly affected by the presence of irradiated plasma which caused a dose-dependent speeding up of the cell cycle.     

Complex cytogenetic characteristic of people suffered from Chernobyl accident

A cytogenetic study was performed on Chernobyl cleanup workers, on their children, on persons evacuated from contaminated aeria (adult and children), on so named "veterans of particular risk" irradiated due to the accidents on the nuclear plant, testing of nuclear weapons etc. and on control donors. The yield of stable (FISH analysis) and of unstable chromosome aberrations, micronuclei in both lymphocytes and erythrocytes, HPRT mutations was found to be increased in exposed groups as compared to control ones. In children of liquidators and in evacuated children we observed genomic instability and increased in vitro chromosomal radiosensitivity. Acceleration of age accumulation of translocations characterized the exposed population in comparison with control group. People with the highest level of routine chromosome aberrations had cardiovascular and digestive diseases more often likely than those with the lowest level. In frame of International Project ECP-6--"Biological dosimetry" the dose-responses for dicentrics and translocations were constructed in dose range 0-100 cGy of gamma-irradiation on the base of data of 8 laboratories. On cancer patients undergone whole-body gamma-irradiation (every day at the dose 11.5 cGy to a total of dose 57.5 cGy) we constructed the dose-responses for the dicentrics and translocations and compared them with the dose-responses for these aberrations after the in vitro irradiation of lymphocytes of the same patients. For the dicentrics the effectiveness of the in vivo irradiation was less than of the in vitro one. No differences were found for translocations.     

Seventeen-year follow-up study on chromosomal aberrations in five victims accidentally exposed to several Gy of <Superscript>60</Superscript>Co γ-rays

On 25 June 1990, a radiation accident occurred in a ⁶⁰Co source radiation unit in Shanghai, due to violations in operation regulations. This accident resulted in the exposure of seven individuals to acute high-dose and dose-rate whole-body external irradiation. Conventional chromosomal aberration analysis, G-banding automatic karyotype analysis and/or fluorescent in situ hybridization (FISH) painting methods were used to analyze chromosomal aberrations in peripheral blood lymphocytes from five of the victims 24 h to 17 years after accidental exposure to 1.9–5.1 Gy of ⁶⁰Co γ-rays. The frequency of unstable chromosomal aberrations (dicentrics and rings) remained at constant levels 1 month after exposure. Three months after exposure, the frequency was reduced by 20–40% in three victims, while no reduction was seen in the other two victims. Twelve years after exposure, the number of dicentrics and rings decreased by more than 90%, and did not reveal a dose-dependent relationship. However, even at 12–17 years after exposure, stable chromosome aberrations, dominated by translocations, remained at a high level in a dose-dependent manner. The frequency of stable chromosomal aberrations detected by FISH showed a similar dose-dependent relationship as that detected by karyotype analysis of G-banding chromosomes. The G-banding analysis also suggested that the pattern of chromosome breakpoints is random. The FISH data showed a decreasing tendency with time for chromosome translocation frequency in the peripheral lymphocytes, and the rate of reduction varied among different individuals. It is likely that the higher dose the victim received, the lesser the translocation frequency decreased with time. The G-banding data also showed that the rate of reduction of translocations is different among individuals. From 5 to 17 years after accidental irradiation, a very small reduction (~10%) of translocation frequency was observed in victims C and D, while there was about a 35% reduction (the highest among the victims) for victim G who received the smallest dose (1.9 Gy). These observations can be used to validate the existence of chromosomal aberrations in peripheral blood lymphocytes as a biological dosimeter for radiation exposures.     

Unstable chromosome aberrations in peripheral blood lymphocytes from patients with cervical uterine cancer following radiotherapy.

Scoring of unstable chromosomes aberrations (dicentrics, rings and fragments) in circulating lymphocytes is the most extensively studied biologic system for estimating individual exposure to ionizing radiation. In this work, blood samples from 5 patients, with cervical uterine cancer, were analyzed by conventional cytogenetic in order to correlate the frequency of chromosome aberrations in lymphocytes with the dose absorbed by the patient, as a result of radiotherapy with 60Co gamma. The samples were collected in three phases of the treatment: before irradiation, 24 hr after receiving 0.08 Gy and 1.8 Gy, respectively. On the basis of the frequencies of unstable aberrations observed, a good agreement was obtained between doses estimated by calibration curve and the doses previously planned to radiotherapy. This report discusses the methodology employed as an important tool for dose assessment as a result of partial-body exposure to ionizing radiation.     

Cytogenetic analysis of peripheral blood lymphocytes of occupational workers exposed to low levels of ionising radiation.

The incidence of chromosomal aberrations was analysed in peripheral blood lymphocytes of occupationally exposed people having cumulative doses of 500 mSv. The exposed individuals showed higher frequencies of dicentrics as well as acentrics than normal controls. Absorbed radiation dose was calculated by using in vitro dose response curve established for Cobalt-60 gamma rays. In the control constituting 17 healthy individuals, two dicentrics were detected among 3700 metaphases analysed. In the exposed group 27 dicentrics and one centric ring was detected among 8400 metaphases analysed. Due to small number of dicentrics scored in each individual, the dose estimate suffers from a large statistical uncertainty. The collective dose was found to be 1.89 Gy. This is in good agreement with the corrected physical doses, assuming a mean life of 10 years for the disappearance of lymphocytes. The physical doses accumulated during the last 10 years of occupation were also in good agreement with the biological dose estimate.     

Radiation-induced chromosomal aberrations in the bone marrow of mice exposed to various doses of gamma radiation

The occurrence of chromosomal aberrations was studied at 1–14 days post-exposure in female BALB/c mice exposed to various doses of gamma radiation. The frequency of abnormal cells, chromatid and chromosome breaks, dicentrics, centric rings, acentric fragments and total aberrations increased with exposure dose, and it was highest at 7 Gy. A peak was recorded on day 1 post-exposure with a gradual decline thereafter. The chromosomal aberration yield reached a nadir on day 14 post-irradiation, without restoration to the control level. The best fit for the present data was by a linear-quadratic relationship between dose of radiation and the frequency of chromosomal aberrations.     

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.     

The cytogenetic effects of 22-MeV neutrons

22-MeV neutrons affecting the culture of human peripheral blood lymphocytes have been studied for their cytogenetic peculiarities. Linear character of the dependence of chromosome aberrations yield within the dose range of 0.2-4.0 Gy is noted. Relative biological efficiency of 22-MeV neutrons reaches the highest values at low doses (18.8-2.4). Pair fragments and dicentrics prevail in spectrum of aberrations.