Analysis of FISH-painted chromosomes in individuals living near the Semipalatinsk nuclear test site.

The Semipalatinsk nuclear test site (STS) is located in the Republic of Kazakhstan. A total of 498 nuclear weapons tests were conducted in this area between 1949 and 1989. The radiation exposure to people who lived close to the STS resulted mostly from the above-ground explosions. Blood samples for chromosome analysis were obtained from 10 subjects who were born before the first explosion in August 1949 and lived continuously in the village of Dolon. The individual calculated effective doses were about 3 Sv. Chromosomes 2, 4 and 8 were painted by means of the FISH technique. In total, 22,240 cells were analyzed. The mean frequency of translocations in the subjects who were irradiated during childhood (2.4/1000 cells) did not differ from the control value (3.1 translocations/1000 cells). It is assumed, therefore, that the calculated physical dose is too high. A significantly increased level of complex cells was determined, however, and this was assumed to have been induced in circulating lymphocytes. The reason for this may be the incorporation of radionuclides from fallout which were not distributed homogeneously within the body, but accumulated instead in tissues that were well supplied with peripheral blood.

Reciprocal translocations as an indicator for radiation exposures in the low dose range.

A few years back, the frequency of dicentrics was determined shortly after exposure in five accidentally exposed radiation workers. In all cases the observed dicentric yield was significantly higher in comparison with the background level, and the resulting estimated whole body doses lay between 0.2 and 0.3 Gy. Now, a number of years later (1 to 11 years), the frequency of translocations has been determined by means of the FISH technique. Chromosomes 2, 4 and 8 were painted. The measured translocation frequency lay, however, within the range of the spontaneous variation between individuals. No radiation exposure could, therefore, be proven. In two further cases, the dicentrics were determined by means of the conventional Giemsa staining technique, and the translocations by means of chromosome painting carried out on the same blood samples, which were taken about 3 and 10 years, respectively, after the radiation exposure. The dose estimates obtained on the basis of dicentric frequency using the Qdr method, and on the basis of FISH detected translocations, are compared.

Involvement of specific chromosomes in radiation-induced rearrangements detected by fluorescence in situ hybridization (FISH).

We investigated the involvement of chromosomes in dicentrics and translocations in human peripheral blood lymphocytes exposed to X-rays in vitro. Chromosomes 2, 4, 8, 13, 15, 16, and 22 were analyzed in three cocktails of different combinations using whole chromosome probes (WCP) with the fluorescence in situ hybridization (FISH)-painting technique. The results showed overexpression of chromosomes 2, 8, and 22 in translocation, the majority being of the complete type. Chromosome 4 was underrepresented in translocation formation in both combinations, that is, 2 + 4 + 8 in cocktail I and 4 + 13 + 22 in cocktail II. Its participation in dicentric production was in good agreement with its DNA content in association with 2, 4, and 8, whereas it was underexpressed in the combination of 4 + 13 + 22. DNA-proportional involvement was noticed with chromosomes 13 and 16 in all exchange aberrations. Underexpression of chromosome 15 was observed in translocation, which is contradictory to its overexpression in dicentric formation. The participation of chromosome 22 was predominant for both translocations and dicentrics, compared with its DNA content. The overall observation of our study supports the assumption of DNA-proportional distribution of Lucas et al. However, more data are required for chromosomes 4, 8, 15, and 22 in combination with other chromosomes.

The influence of age, sex and smoking habits on the background level of fish-detected translocations.

Chromosomal aberrations have been used for biological dosimetry for many years but dicentric yield decreases with time after irradiation. Since translocation yields should persist in peripheral lymphocytes with time they should prove to be a better indicator in the detection of old and chronic exposures to ionising radiations. The precondition, however, is knowledge of the control level in healthy subjects exposed only to normal background and also knowledge of confounding factors. From 42 healthy subjects, aged between 21 and 73 years, about 120,000 FISH-painted cells were scored using DNA probes for chromosomes 2, 4 and 8, and an all-human centromere probe. The statistical analyses revealed no influence of gender on the translocation frequency. Smoking habits-even a daily consumption of >30 cigarettes-seem to have only a marginal impact on the translocation yield, whereas an age-dependence has been established. Because of the high background level of translocations and the variation in the frequency between subjects, the lowest detectable limit for radiation exposures should be between 0.3 and 0.5 Gy, assuming that blood stem cells have a similar radiation sensitivity to that of peripheral lymphocytes for the induction of translocations.

Chromosomal aberrations in peripheral lymphocytes from healthy subjects as detected in first cell division.

Baseline frequencies of chromosomal aberrations were analysed in human peripheral lymphocytes and the influence of age, sex and smoking habits was considered. From 53 healthy subjects (29 males, 24 females) 54,689 exclusively first division cells (M1) were scored. The frequencies of chromosome aberrations per 1000 cells were 1.15 +/- 0.15 dicentrics (dic), 2.6 +/- 0.3 excess acentric fragments (ace) and 7.0 +/- 0.6 chromatid breaks (crb). An age dependency could only be established for ace. Between males and females no differences in any of the aberration types were observed. For heavy smokers (> 30 cigarettes per day) a significant increase was only found for dic (2.5 +/- 0.6 per 1000 cells). Dicentric frequency was compared with background levels of other studies in which results were reported also from exclusively M1 cells. Despite cell cycle control, differences between laboratories can be observed which may be partly influenced by environmental conditions. But on the other hand the mean frequency of dic (excluding heavy smokers) of 0.95 per 1000 cells reported here is consistent for more than one decade. Since such a consistency of the mean frequency of dic is reported also from another laboratory, the conclusion is drawn that especially for the detection of low-level exposures, each laboratory should establish its own base line data, otherwise, the interpretation of the findings is dependent on the selected background level from the literature.

Analysis of symmetrical translocations for retrospective biodosimetry in radiation workers of the Mayak nuclear-industrial complex (Southern Urals) using FISH-chromosome painting.

PURPOSE: Frequencies of symmetrical translocations were determined by fluorescence in situ hybridization (FISH) for retrospective biodosimetry in workers occupationally exposed to external gamma-rays and internal plutonium at the Mayak nuclear-industrial complex (Southern Urals, Russia). MATERIALS AND METHODS: Chromosome analyses were carried out on peripheral lymphocytes from 75 Mayak workers who had received their main exposures between 1948 and 1963. Cumulative external gamma-ray doses between 0.02 and 9.91 Sv and plutonium burdens ranging between 0.26 and 18.5 kBq are reported. As controls, 33 unexposed persons from non-contaminated areas of the Southern Urals were used. Whole-chromosome painting probes for chromosomes 1, 4 and 12 were used simultaneously with a pancentromeric probe. RESULTS: Compared with the control group, a significantly elevated translocation frequency was found for the total study group and for either of two subsets with (48 subjects) and without (27 subjects) plutonium incorporation. The dicentric frequency was not significantly different from the control level. In the pooled data set, translocation frequencies showed a significant dependence on cumulative external gamma-ray doses. Plutonium uptake had no substantial influence. Individual dose estimates for 21 cases exhibiting at least five translocations ranged between 0.5 and 1.8 Gy, which is substantially lower than the workers' registered personal doses. CONCLUSION: At 35-40 years after protracted exposure to low-dose rate external gamma-rays, the postulated lifetime stability of translocations cannot be confirmed. Apparently, the natural loss of translocation-bearing peripheral lymphocytes cannot be fully compensated so that a temporal decline even of transmissible aberrations takes place. As a consequence, individual retrospective biodosimetry estimates cannot be obtained reliably from the remaining fraction of translocations.

Chromosome translocations detected by fluorescence in situ hybridisation (FISH)--a useful tool in population monitoring?

It is assumed that symmetrical translocations persist in peripheral lymphocytes for years. For this reason, they should be valuable indicators especially for chronic exposures of populations to mutagens or carcinogens. Reciprocal translocations can be easily detected by means of fluorescence in situ hybridization (FISH). Very little is known about the spontaneous frequency, which forms the basis for population monitoring, and the variations which exist between individuals. The frequencies obtained on the basis of structural chromosome aberrations detected by means of FISH in 29 healthy persons are presented, and the deviations in the occurrence of symmetrical translocations arising in the different persons are indicated. Tests for the determination of persistence levels, are carried out on persons exposed to radiation. The chromosomes 2, 4 and 8 are painted parallel to the centromeres, by means of the FISH technique.

FISH-based analysis of stable translocations in a Techa River population.

PURPOSE: Measurements of symmetrical translocations by fluorescence in situ hybridization (FISH) were performed for retrospective biodosimetry in a Techa River population exposed to external (gamma-rays) and internal (90Sr, 137Cs) irradiation. MATERIALS AND METHODS: Chromosome analyses were carried out on peripheral lymphocytes from 73 radiation-exposed residents from settlements along the Techa River (Southern Urals, Russia) located 7-148 km downstream from the site of release of liquid radioactive waste from the plutonium production facility Mayak. Thirty-nine unexposed persons from non-contaminated areas were used as controls. Whole-chromosome painting probes for chromosomes 1, 4 and 12 were used simultaneously with a pancentromeric probe. RESULTS: A significantly elevated mean translocation frequency compared with controls was found for the total study group and for either of two subgroups of inhabitants residing in villages of the upper regions of the Techa River (7-60 km) during 1950 to 1951, or in villages of the lower regions (78-148 km) until the time of blood sampling. Within the first subgroup, subjects born between 1937 and 1949 showed higher translocation frequencies than those born between 1914 and 1936. Collective biodosimetry estimates for the various groups were between 0.24 and 0.54 Gy. Individual dose estimates for seven subjects with at least five translocations ranged between 0.77 and 1.80 Gy and compared well with doses reconstructed on the basis of 90Sr whole-body counts (WBC) and electronic paramagnetic resonance (EPR) measurements. Individual translocation frequencies from 40 subjects with existing WBC data and calculated cumulative red bone marrow doses below 0.6 Gy fall within the 95% prediction limits of the calibration curve. CONCLUSIONS: FISH-based translocation measurements can provide useful information for a retrospective biodosimetric interpretation. However, with the analysed number of cells, individual estimates required for a reliable evaluation of this highly variable exposure situation can be obtained only for a minority of subjects. In addition, influence of a temporal decline on the yield of stable translocations cannot be fully excluded.

Chromosome aberrations in human lymphocytes analysed by fluorescence in situ hybridization after in vitro irradiation, and in radiation workers, 11 years after an accidental radiation exposure.

Fluorescence in situ hybridization of metaphase chromosomes was used to determine the yield of symmetric and asymmetric exchange aberrations after in vitro exposure of peripheral lymphocytes to 250 kV X-rays (0-3.0 Gy). For the aberration analyses, chromosomes 2, 4 and 8 and all centromeres were painted. Centric rings amounted to about 8% of the dicentric yield. The proportion of inversions and insertions was about 5% of the total translocations. Regarding the spontaneous levels, the frequency of total induced translocations was higher by a factor of 1.13 than that of dicentrics. The involvement of chromosomes 2, 4 and 8 in translocations is significantly different from the expected ratio concerning physical length (p < 0.01). Furthermore, the frequency of translocations was evaluated in three radiation workers who received an accidental radiation exposure 11 years ago. About 75% of the translocations were identified as complete in comparison with 79% in the in vitro experiments. In the radiation workers chromosome 2 again showed an under-representation in translocations, whereas chromosome 4 was over-represented as in the in vitro experiments. The summarized results for the radiation workers showed a mean genomic translocation frequency of 13.4 per 1000 cells. This frequency is not significantly different from the mean frequency of dicentrics which were determined by conventional FPG staining, after detection of the accidental radiation exposure about 11 years ago (8.6 dic/1000 cells). There were, however, some differences between individuals affecting this comparison. The distribution patterns of dicentrics showed an over-dispersion, whereas the translocations occurred single in cells.

Combined immunophenotyping and interphase cytogenetics on cryostat sections by the new FICTION method.

A technique is described to detect tumor cells with certain chromosome aberrations within cryostat sections and to characterize these individual cells by immunophenotyping. This new method is called fluorescence-immunophenotyping and interphase cytogenetics as a tool for investigation of neoplasms (FICTION).