Effect of age and radiation exposure on the frequency of translocations and dicentrics detected by FISH method in human lymphocytes

The frequencies of translocations and dicentrics detected by "chromosome painting" in lymphocytes were estimated in 115 healthy donors and in 273 people exposed to uncontrolled irradiation at low doses 1-4 years ago. Age responses of both types of exchanges at the age range from 3 to 85 years fit to quadratic model. The frequency of translocations grew faster with age than the frequency of dicentrics. The yields of stable exchanges in exposed people was significantly higher than those in control donors of corresponding ages.     

Stable and unstable chromosome aberrations in humans and other mammals in relation to the problems of biological dosimetry

Literature data of long-term cytogenetic follow-up of people exposed to radiation as a results of different radiation accidents are considered for the purpose of discussing of some problems of biological dosimetry. The results obtained for mammals are also presented. Of particular interest is a decrease in the level of dicentrics and symmetrical translocations in peripheral blood lymphocytes with the time after acute exposure depending on the dose of irradiation. The frequency of dicentrics decreases in accordance to the exponential law passing the fast and slow phases of elimination. In different radiation situations the values of the parameter which defines the half-life period of lymphocytes characterizing 50% reduction of cells with dicentrics markedly vary. However a general regularity is a decrease in the parameter value as the exposure dose increases. The level of stable translocations estimates by the EISH method remains relatively constant at doses below 1-2 Gy. At higher doses their level in peripheral blood lymphocytes declines with time due to which the retrospective dose appears to be underestimated. The reasons of such regularity, the role of various factors affecting the scoring of translocations, criteria of analysis of the given chromosome aberrations are discussed in the context of common agreements between leading European laboratories on the use of FISH for improving biological dosimetric estimates.     

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.     

Choosing metaphases for biological dosimetry by fluorescence in situ hybridization (FISH)

Data are presented for a subset of lymphocytes characterized by FISH as missing painted chromosomal material. These lymphocytes occur in both control and irradiated subjects. These cells have a much greater frequency of one-way translocations than cells in which all of the painted chromosomal material is present. Their presence contributes to interindividual variability in control translocation yields. These cells do not appear to be more prevalent in persons exposed to high radiation doses. It is suggested that their exclusion when selecting cells for analysis may improve the sensitivity of FISH as a biological dosimeter at low doses. Mechanisms for the production of these one-way translocations in vivo are also discussed, with a proposal that their variable frequency in individuals may be consistent with exposure to chemical clastogens.     

Retrospective dose estimation in remote period after exposure using different biological methods

Investigation of application of chromosome aberrations of lymphocytes in peripheral blood for biological dosimetry purposes in remote (up to 40 years) period after acute exposure to doses of 1 Gy and more was carried out. The comparative analysis of frequency of unstable and stable (using FISH and G-banding methods) aberrations was performed for 24 subjects accidentally exposed to radiation on nuclear submarines during 1961-1985. Statistically significant increasing of frequency of dicentrics and centric rings was determined in the exposed subjects in remote period after exposure to compare with controls. Their sum frequency in the exposed group varied depending on ARS heaviness from 0.1 to 1.0 aberrations per 100 cells. In control group it was from 0 to 0.2 correspondingly. Translocation frequency (complete + incomplete) fixed by FISH method (2, 4, and 12 chromosomes) varied within the limits of 0.2-16.0 for exposed subjects and 0.3-1.26 translocations per genome per 100 cells for controls. Some examined persons (5 subjects) exposed to accident in 1985 had results of analysis of unstable chromosome aberration in acute period after exposure that allow to estimate obtained doses by dicentrics frequency which having good correlation with ARS heaviness. Individual dosed using traslocation frequency were defined retrospectively in 11 from 21 exposed persons. They correlate with calculated physics doses and doses estimated by haematolotical parameters in acute period and also doses obtained by ESR spectroscopy of tooth enamel in remote period.     

A cytogenetic follow-up study of the victims of a radiation accident in Goiania (Brazil)

A radiation accident involving a cesium-137 therapy source occurred in Goiania (Brazil) in September 1987, in which more than 50 individuals were exposed to moderate to high doses (0.2-7 Gy) of gamma-radiation. A cytogenetic technique (i.e., frequencies of dicentrics and rings in peripheral lymphocytes) was employed to estimate the absorbed radiation dose. The follow-up study extending over more than 1 year indicated a decline in the frequencies of dicentrics in the lymphocytes. Using chromosome-specific biotinylated library probes for chromosomes 1, 2, 8 and 19, we studied the frequencies of chromosomal translocations and deletions and the incidence of aneuploidy in the lymphocytes of exposed individuals. In some individuals there was a significant increase in the frequency of translocations and aneuploidy. In other experiments, in which the frequencies of HPRT mutations were determined in lymphocytes using the BrdU-labeling method, some individuals showed an increase (from about 2- to 50-fold) in mutant frequencies.     

Induction of chromatid-type aberrations in peripheral lymphocytes of hospital workers exposed to very low doses of radiation

Radiological personnel represent workers exposed to low cumulative doses of radiation. As their surveillance is generally based on physical dosimetry, there is little or inconclusive information on biological effects due to radiation exposure at these doses. We aimed to explore the extent of chromosomal damage in circulating lymphocytes of hospital workers (technicians, nurses and physicians) chronically exposed to a very low level of radiation using conventional and molecular cytogenetic analyses (chromosome painting with chromosomes #2, #3 and #10 as probe cocktail). Compared with controls, exposed workers displayed a significant increase in the frequency of aberrant lymphocytes (1.26+/-0.11/100 cells versus 1.63+/-0.17/100 cells). In particular, exposed technicians showed significantly higher mean values than nurses or physicians (3.68+/-1.17/100 cells versus 1.36+/-0.18/100 cells and 1.36+/-0.09/100 cells, respectively). Interestingly, we found that the chromosomal damage was prevalently expressed as chromatid-type aberrations. Chromosome painting indicated that the frequency of chromosome rearrangements (CR; translocations and dicentrics pooled together) was approximately comparable between radiological workers and the control group. Moreover, we did not detect any significant difference due to radiation exposure when CR rates were considered separately for each of the three chromosomes in the probe cocktail.     

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.     

Biological dosimetry in a group of radiologists by the analysis of dicentrics and translocations

The results of a cytogenetic study carried out in a group of nine radiologists are presented. Chromosome aberrations were detected by fluorescence plus Giemsa staining and fluorescence in situ hybridization. Dose estimates were obtained by extrapolating the yield of dicentrics and translocations to their respective dose-effect curves. In seven individuals, the 95% confidence limits of the doses estimated by dicentrics did not include 0 Gy. The 99 dicentrics observed in 17,626 cells gave a collective estimated dose of 115 mGy (95% confidence limits 73-171). For translocations, five individuals had estimated doses that were clearly higher than the total accumulated recorded dose. The 82 total apparently simple translocations observed in 9722 cells gave a collective estimated dose of 275 mGy (132-496). The mean genomic frequencies (x100 +/- SE) of complete and total apparently simple translocations observed in the group of radiologists (1.91 +/- 0.30 and 2.67 +/- 0.34, respectively) were significantly higher than those observed in a matched control group (0.53 +/- 0.10 and 0.87 +/- 0.13, P < 0.01 in both cases) and in another occupationally exposed matched group (0.79 +/- 0.12 and 1.14 +/-0.14, P < 0.03 and P < 0.01, respectively). The discrepancies observed between the physically recorded doses and the biologically estimated doses indicate that the radiologists did not always wear their dosimeters or that the dosimeters were not always in the radiation field.     

High susceptibility of chromosome 16 to radiation-induced chromosome rearrangements in human lymphocytes under in vivo and in vitro exposure

The aim of the present study was to investigate whether chromosome 16p presents breakpoint regions susceptible to radiation-induced rearrangements. The frequencies of translocations were determined by fluorescence in situ hybridization (FISH) using cosmid probes C40 and C55 mapping on chromosome 16p, and a chromosome 16 centromere-specific probe (pHUR195). Peripheral lymphocytes were collected from normal individuals and from seven victims of 137Cs in the Goiania (Brasil) accident (absorbed doses: 0.8-4.6 Gy) 10 years after exposure. In vitro irradiated lymphocytes (3 Gy) were also analyzed. The mean translocation frequency/cell obtained for the 137Cs exposed individuals was 2.4-fold higher than the control value (3.6 x 10(-3) +/- 0.001), and the in vitro irradiated lymphocytes showed a seven-fold increase. The genomic translocation frequencies (FGs) were calculated by the formula Fp = 2.05 fp(1-fp)FG (Lucas et al., 1992). For the irradiated lymphocytes and victims of 137Cs, the FGs calculated on the basis of chromosome 16 were 2- to 8-fold higher than those for chromosomes 1, 4 and 12. Our results indicate that chromosome 16 is more prone to radiation-induced chromosome breaks, and demonstrate a non-random distribution of induced aberrations. This information is valuable for retrospective biological dosimetry in case of human exposure to radiation, since the estimates of absorbed doses are calculated by determining the translocation frequency for a sub-set of chromosomes, and the results are extrapolated to the whole genome, assuming a random distribution of induced aberrations. Furthermore, the demonstration of breakpoints on 16p is compatible with the reports about their involvement in neoplasias.     

Frequency of spontaneous chromosome aberrations in mice: effects of age

In this paper, we present data on chromosome aberration frequencies in mice which served as unexposed controls in a variety of radiation and chemical toxicology experiments conducted in our laboratory in recent years. All chromosome aberration data were obtained by chromosome painting. In peripheral blood lymphocytes from 102 animals, the frequencies of translocations and insertions increased significantly with age. No increase with age was seen for dicentrics or acentric fragments. When the data were analyzed by strain, the age-related increase in translocation frequencies was observed only in the 71 homozygous C57BL/6 mice and not in any of the three heterozygous strains. Very few aberrations of any type were observed in 62 bone marrow samples, and no effect of age was seen for any aberration type in this tissue. These results are similar to those observed in unexposed humans, and suggest that the increase in translocations is not the result of accumulated damage from chronic 'background' environmental exposures but instead may be due to biological processes associated with aging.     

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.     

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.     

Possibilities and limitations of fluorescence in situ hybridization technique in retrospective detection of low dose radiation exposure in post-chernobyl human cohorts

Cytogenetic analysis using the fluorescence in situ hybridisation (FISH) technique was performed late time after the Chernobyl accident in groups of liquidators, evacuees from 30 km exclusive zone, residents of radioactively contaminated areas and control donors age-matched to exposed persons. Stable and unstable chromosome type exchanges were recorded using a hybrid conventional-PAINT nomenclature. The mean yield of stable chromosome exchanges in liquidators did not correlate with registered radiation doses but had a clear negative dependence on the duration of liquidators' staying in Chernobyl zone, that was in a good agreement with early data based on conventional dicentrics plus rings analysis. The overspontaneous excess for stable chromosome exchange level appeared to be higher in evacuees 16-40 years old than that of senior persons, whereas no age-dependent difference occurred for initially induced dicentrics plus rings yields in this cohort. The stable chromosome exchange yield, as well as combined yield of dicentrics plus rings and potentially unstable incomplete translocations in residents of radioactively contaminated areas showed a reasonable positive correlation with levels of 137Cs contamination. The observed yields of stable chromosome exchanges in all three exposed groups appeared to be somewhat lower than those of expected from unstable exchange-based doses which were referred to an in vitro dose response of stable exchanges outcome in human lymphocytes. Thus, FISH analysis can be successfully applied for qualitative cytogenetic indication of past and chronic radiation exposure to low doses but further refinement of FISH-based system for quantitative dose assessment is still required. Some practical approaches of solving this task are discussed.     

Low-dose ionizing radiation and chromosome translocations: a review of the major considerations for human biological dosimetry

Chromosome translocations are a molecular signature of ionizing radiation exposure. Translocations persist significantly longer after exposure than other types of chromosome exchanges such as dicentrics. This persistence makes translocations the preferred aberration type for performing radiation dosimetry under conditions of protracted exposure or when exposure assessments are temporally delayed. Low doses of radiation are inherently difficult to quantify because the frequency of induced events is low and the background level of translocations among unexposed subjects can show considerable variability. Analyses of translocation frequencies can be confounded by several factors, including age of the subject, lifestyle choices such as cigarette smoking, the presence of clones of abnormal cells, and possibly genotypic variability among subjects. No significant effects of gender or race have been observed, but racial differences have not been completely ruled out. Translocation analyses may be complicated by the presence of different types of exchanges, i.e., reciprocal or non-reciprocal, and because translocations sometimes occur as a component of complex exchanges that include other forms of chromosome rearrangements. Rates of radiation exposure, ranging from acute to chronic, are known to influence the accumulation of translocations and may also affect their persistence. The influences on translocation frequencies of low-dose radiation hypersensitivity as well as the bystander effect and the adaptive response remain poorly characterized. Thus, quantifying the relationship between radiation dose and the frequency of translocations in any given subject requires attention to multiple issues. Part of the solution to understanding the in vivo dose-response relationship is to have accurate estimates of the baseline levels of translocations in healthy unexposed subjects, and some work in this area has been accomplished. Long-term cytogenetic follow-up of exposed subjects is needed to characterize translocation persistence, which is especially relevant for risk analyses. More work also needs to be done in the area of quantifying the role of known confounders. Characterizing the role of genotype will be especially important. Improvements in the ability to use translocation frequencies for low-dose biological dosimetry will require scoring very large numbers of cells per subject, which may be accomplished by developing a rapid automated image analysis system. This work would enhance our comprehension of the effects of low-dose radiation exposure and could lead to significant improvements in understanding the relationship between chromosome damage and human health.     

Three somatic genetic biomarkers and covariates in radiation-exposed Russian cleanup workers of the chernobyl nuclear reactor 6-13 years after exposure

Three somatic mutation assays were evaluated in men exposed to low-dose, whole-body, ionizing radiation. Blood samples were obtained between 1992 and 1999 from 625 Russian Chernobyl cleanup workers and 182 Russian controls. The assays were chromosome translocations in lymphocytes detected by FISH, hypoxanthine phosphoribosyltransferase (HPRT) mutant frequency in lymphocytes by cloning, and flow cytometic assay for glycophorin A (GPA) variant frequency of both deletion (N/?) and recombination (N/N) events detected in erythrocytes. Over 30 exposure and lifestyle covariates were available from questionnaires. Among the covariates evaluated, some increased (e.g. age, smoking) and others decreased (e.g. date of sample) biomarker responses at a magnitude comparable to Chernobyl exposure. When adjusted for covariates, exposure at Chernobyl was a statistically significant factor for translocation frequency (increase of 30%, 95% CI of 10%-53%, P = 0.002) and HPRT mutant frequency (increase of 41%, 95% CI of 19%-66%, P < 0.001), but not for either GPA assay. The estimated average dose for the cleanup workers based on the average increase in translocations was 9.5 cGy. Translocation analysis is the preferred biomarker for low-dose radiation dosimetry given its sensitivity, relatively few covariates, and dose-response data. Based on this estimated dose, the risk of exposure-related cancer is expected to be low.     

The cellular lethality of radiation-induced chromosome translocations in human lymphocytes

Recent evidence has shown that translocation frequencies decline over time. This phenomenon might be explained by the co-occurrence of translocations in cells that also contain dicentrics, in which case translocations would be eliminated as a by-product of selection against dicentrics. Alternatively, a fraction of translocations may themselves be lethal. Here we describe our initial approaches to develop mathematical models to test whether the decline in translocation frequencies results from the first, the second, or a combination of these two possibilities. The models assumed that all chromosome exchanges were simple, i.e., were comprised of dicentrics as well as one-way and two-way translocations. Complex aberrations (three or more breaks in two or more chromosomes) were not modeled, nor were fragments or intrachromosomal exchanges (rings, inversions). We tested the models using Monte Carlo simulations, and then we fitted the models to data describing chromosome aberration frequencies induced by a single acute in vitro exposure to (137)Cs gamma rays in human peripheral blood lymphocytes from two donors. Chromosome painting was used to enumerate translocations and dicentrics from 2 to 7 days after exposure. Our results indicate that in donor no. 2, the decline in translocation frequencies occurs as a by-product of selection against dicentrics. However, in donor no. 1, whose cells appeared more radiosensitive than cells from donor no. 2, up to 40% of the one-way translocations may themselves be lethal at high doses, although calculations indicate that two-way translocations do not cause lymphocyte mortality. Individual variation in the probability that translocations are lethal to cells appears to be important, and one-way translocations appear to be lethal more often than two-way translocations. Within the limits of these models, these findings indicate that both postulated mechanisms, i.e. inherent lethality and selection against dicentrics in the same cells, contribute to the loss of both one-way and two-way translocations.     

Effect of high-level natural radiation on chromosomes of residents in southern China

To study the effect of low-dose (rate) radiation on human health, we analyzed chromosomes of peripheral lymphocytes of residents in a high background radiation area (HBRA) and compared the results with those obtained from residents in a control area (CA) in Guangdong Province, China. Unstable types of chromosome aberrations (dicentrics and rings) were studied in 22 members of eight families in HBRA and 17 members of five families in CA. Each family consists of three generations. On average 2,600 cells per subject were analyzed. 27 adults and six children in HBRA and 25 adults and eight children in CA were studied with respect to translocations. On average 4,741 cells per subject were examined. We found an increase of the frequency of dicentrics and rings in HBRA, where the natural radiation level is three to five times higher than in the control area. But the increase of translocations in HBRA was within the range of individual variation in the controls.     

Cesium-induced chromosome aberrations analyzed by fluorescence in situ hybridization: eight years follow up of the Goiânia radiation accident victims

The radiation accident in focus here occurred in a section of Goiânia (Brazil) where more than a hundred individuals were contaminated with ¹³⁷Cesium on September 1987. In order to estimate the absorbed radiation doses, initial frequencies of dicentrics and rings were determined in 129 victims [A.T. Ramalho, PhD Thesis, Subsidios a tecnica de dosimetria citogenetica gerados a partir da analise de resultados obtidos com o acidente radiologico de Goiânia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil, 1992]. We have followed some of these victims cytogenetically over the years seeking for parameters that could be used as basis for retrospective radiation dosimetry. Our data on translocation frequencies obtained by fluorescence in situ hybridization (FISH) could be directly compared to the baseline frequencies of dicentrics available for those same victims. Our results provided valuable information on how precise these estimates are. The frequencies of translocations observed years after the radiation exposure were two to three times lower than the initial dicentrics frequencies, the differences being larger at higher doses (> 1 Gy). The accuracy of such dose estimates might be increased by scoring sufficient amount of cells. However, factors such as the persistence of translocation carrying lymphocytes, translocation levels not proportional to chromosome size, and inter-individual variation reduce the precision of these estimates.     

The significance of cytogenetic investigation for the estimation of Chernobyl accident consequences

The results of the cytogenetic investigation of people, which were exposed to radiation in the result of the Chernobyl NPP accident, were presented. Also the possibilities of the application of cytogenetic findings for dose estimations and for the prediction of the radiation influence consequences were examined. During the period of time since 1986 till 2004 the cytogenetic investigations of 1724 liquidators participating in the liquidation works after the Chernobyl accident were carried out. The radiation dose estimated by the frequency of dicentrics in 1986 was about 0.16 Gy. The doses for liquidators were determined by the frequency of translocations (FISH method) during the period from 1992 till 1995. For liquidators who worked in Chernobyl only in 1986 the average dose of radiation was about 0.19 Gy and for liquidators who worked repeatedly during the period from 1986 till 1995 - 0.39 Gy. There was shown that during the whole period of investigation (1986-2004) the frequency of dicentrics in peripheral blood lymphocytes was significantly higher than the control level. The cytogenetic investigation of Bryansk region inhabitants which was carried out in 1992-1994 discovered heightened value in 5 times than the control one. Findings are of great importance for the prediction of ill effects of radiation and for the development of sensitive criterions for early exposure disturbances in state of health.