Evaluation of three somatic genetic biomarkers as indicators of low dose radiation effects in clean-up workers of the Chernobyl nuclear reactor accident.

The goals of this study were to assess three biomarkers of genetic effect for their individual and collective ability to detect and estimate radiation exposure in Russian Chernobyl clean-up workers. Work assignments were planned to limit dose to 0.25 Gy. The three biomarkers employed were chromosome translocations detectcd in lynmphocytes by florescence in situ hybridisation (FISH), and mutation at two genes, glycophorin A (GPA) in red blood cells detected by flow cytometry and hypoxanthine phosphoribosyltransferase (HPRT) in lymphocytes detected by selective cell culture. Samples were Obtained from 1992 to 2000. The time between exposure at Chernobyl and sample acquisition was > or =5 years. The lymphocyte assays detected an elevation over controls in average outcomes it clean-up workers: translocation rates were 46% higher when adjusted for age and smoking and HPRT mutant frequencies were were 16% higher when adjusted for age. The G PA assay did not detect an exposure effect. The results indicate that measuring frequency of translocations by FISH is preferred for low dose radiation, retrospective biochemistry.

[Effect of age and radiation exposure on the frequency of translocations and dicentrics detected by FISH method in human lymphocytes]. / Vliianie vozrasta i oblucheniia na chastotu translokatsii i ditsentrikov, opredeliaemykh metodom FISH, v limfotsitakh cheloveka.

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.

[Concentration of heavy elements and their compounds in the cells from BALF and pulmonary tumors in radiation-exposed subjects]. / Soderzhanie tiazhelykh élementov i ikh soedinenii v kletkakh bronkhoaal'veoliarnykh smyvov i v tkaniakh opukholei legkikh u lits, podvergavshikhsia radiatsionnomu vozdeistviiu.

AIM: Measurement of heavy elements in alveolar macrophages (AM) of bronchoalveolar lavage (BAL) and pulmonary tumor tissue (PTT); investigation of spacial distribution of the heavy particles in the tissues. MATERIALS AND METHODS: Laser mass-spectrometry estimated quantitative and qualitative characteristics of heavy elements (Mn, Fe, Co, Cu, Zr, etc.) in diagnostic and therapeutic BAL of 6 patients exposed to radiation after the Chernobyl accident and in pulmonary tumor tissue of patients operated for central small-cell and peripheral bronchoalveolar cancer who also had been exposed to radiation after the Chernobyl. RESULTS: Heavy elements concentration in the secondary BAL was less than in the primary one. This shows effectiveness of the procedures aimed at removal of heavy particles from the respiratory organs. Heavy elements content was quite different in two histologically different tumors. CONCLUSION: Laser mass-spectrometry proved its usefulness in simultaneous study of the quantity and quality of heavy elements in AM of BAL and tumors in radiation-exposed subjects, in investigation of the elements distribution in the pulmonary tissues and changes of elements composition in the tissues in the course of tumors development.

Elevated frequencies of hypoxanthine phosphoribosyltransferase lymphocyte mutants are detected in Russian liquidators 6 to 10 years after exposure to radiation from the Chernobyl nuclear power plant accident.

This study was conducted to determine whether the frequency of hypoxanthine phosphoribosyltransferase (HPRT) deficient lymphocyte mutants would detect an effect of radiation exposure in a population of Russians who were exposed to low levels of radiation while working in 1986 and 1987 as liquidators cleaning up after the Chernobyl nuclear power reactor accident. The HPRT lymphocyte cloning assay was performed on peripheral blood lymphocytes collected between 1992 and 1996 from 142 liquidators and 66 Russian controls, and between 1989 and 1993 from 231 American controls. Russian and American controls were not significantly different for either cloning efficiency or mutant frequency (MF); inclusion of both sets of controls in the analysis increased the ability to detect a Chernobyl exposure effect in the liquidators. After adjusting for age and smoking, the results revealed no significant difference in cloning efficiency of Chernobyl liquidators relative to Russian controls but a significant, 24% increase in liquidator HPRT mutant frequency over Russian controls (90% confidence interval was 7% to 45% increase). The analytical method also accounted for differences in precision of the individual estimates of log CE and log MF and accommodated for outliers. The increase in HPRT mutant frequency of liquidators is an attribute of the exposed population as a whole rather than of individuals. These results demonstrate that, under appropriate circumstances, the HPRT specific locus mutation assay of peripheral blood lymphocytes can be used to detect a semi-acute, low dose radiation exposure of a population, even 6 to 10 years after the exposure.

Total gene deletions and mutant frequency of the HPRT gene as indicators of radiation exposure in Chernobyl liquidators.

This study was conducted to determine the utility of deletion spectrum and mutant frequency (MF) of the hypoxanthine phosphoribosyl transferase gene (HPRT) as indicators of radiation exposure in Russian Liquidators who served in 1986 or 1987 in the clean up effort following the nuclear power plant accident at Chernobyl. HPRT MF was determined using the cloning assay for 117 Russian Controls and 122 Liquidators whose blood samples were obtained between 1991 and 1998. Only subjects from whom mutants were obtained for deletion analysis are included. Multiplex PCR analysis was performed on cell extracts of 1080 thioguanine resistant clones from Controls and 944 clones from Liquidators. Although the deletion spectra of Liquidators and Controls were similar overall, the Liquidator deletion spectrum was heterogeneous over time. Most notable, the proportion of total gene deletions was higher in 1991-1992 Liquidators than in Russian Controls (chi 2 = 10.5, p = 0.001) and in 1993-1994 Liquidators (chi 2 = 8.3, p = 0.004), and was marginally elevated relative to 1995-1996 Liquidators (chi 2 = 3.3, p = 0.07). This type of mutations has been highly associated with radiation exposure. Total gene deletions were not increased after 1992. Band shift mutations were also increased in the 1991-1992 Liquidators but were associated with increased MF of both Liquidators and Controls (p = 0.009), not with increased MF in 1991-1992 Liquidators (p = 0.7), and hence are not believed to be associated with radiation exposure. Regression analysis demonstrated that relative to Russian Controls HPRT MF was elevated in Liquidators overall when adjusted for age and smoking status (37%, p = 0.0001), and also was elevated in Liquidators sampled in 1991-1992 (72%, p = 0.0076), 1993-1994 (22%, p = 0.037), and 1995-1996 (62%, p = 0.0001). In summary, HPRT MF was found to be the more sensitive and persistent indicator of radiation exposure, but the specificity of total gene deletions led to detection of probable heterogeneity of radiation exposure within the exposed population.

[Cytological characteristics, elemental composition of alveolar macrophages from bronchoalveolar lavage, lung pathology in liquidators of the consequences of the accident at the Chernobyl power plant]. / Tsitologicheskie osobennosti, élementnyi sostav al'veoliarnykh makrofagov bronkhoal'veoliarnykh smyvov, patomorfologiia legkikh u likvidatorob posledstvii avarii na Chernobyl'skoi AES.

Bronchoalveolar lavage fluid (BALF), broncho- and transbronchial biopsies were examined in Chernobyl wreckers eleven years after the accident. Cytologically and electron-microscopically, there was predominance of alveolar macrophages (AM) with many cytoplasmic foreign bodies containing clear needle-like crystals. Roentgenospectral analysis and laser mass-spectrometry revealed chemical elements which are part of non-active "hot particles" (Al, Si, Ca, Fe) as well as reactor fuel elements and the station constructive materials which are the active part of "hot particles". Programmed BALF allowed to decrease cytosis and the amount of AM containing foreign-body particles as well as concentration of the majority of chemical elements in AM.

[The estimation of the level of mutations at the glycophorin A locus in subjects exposed to the chronic action of ionizing radiation]. / Otsenka urovnia mutatsii v lokuse glikoforina A u lits, podvergavshikhsia khronicheskomu vozdeistviiu ioniziruiushchei radiatsii.

11 persons, who had been irradiated chronically at low dose rate under occupational conditions in 1950s in doses 220-581 cGy according data of individual film dosimeters, and 5 control persons were examined regarding the level of glycophorin A (GPA) mutation type NO and NN in blood erythrocytes. Significantly higher level of GPA mutations type NO was registered in average in the group of exposed persons (23.2 +/- 4.6 x 10(-6)) compared with the control group (10.2 +/- 2.1 x 10(-6)) through the dose dependence was expressed slightly. The coefficient of the linear regression has equaled (2.3 +/- 1.2 x 10(-6)) Gy. The outlook on GPA assay usage in retrospective biodosimetry is discussed.

A study of the effects of exposure on cleanup workers at the Chernobyl nuclear reactor accident using multiple end points.

Blood samples were collected from 192 exposed workers who participated in the cleanup after the April 26, 1986, nuclear reactor accident at Chernobyl, Ukraine. These samples, together with samples from 73 individuals living in Russia but not involved in Chernobyl cleanup activities, were collected during September 1991 to May 1996 and shipped to the U.S. for evaluation by three bioassays: cytogenetic analysis based on chromosome painting, HPRT mutation analysis and glycophorin A (GPA) variant analysis. Univariate statistical analyses of the results of each bioassay (including adjustments for age, smoking status and estimated precision of the bioassay) found greater frequencies of chromosome translocations and HPRT mutant T lymphocytes among the exposed individuals compared to the controls (P < or = 0.01). GPA analyses showed no significant difference for exposed compared to controls for either hemizygous, N/O, or homozygous, N/N, variant cell frequency. Multivariate analysis of variance of the subset of 44 exposed and 14 unexposed individuals with measurements from all three bioassays found elevated frequencies of chromosomal translocations and HPRT mutants, and reduced frequencies for both GPA end points among the exposed persons compared to the controls. However, none of these differences, considered singly or in combination, was statistically significant (although statistical power is low due to small sample sizes). Mean estimated dose, based on cytogenetic response, for those exposed was 9 cGy (range 0 to 51 cGy) and was less than that estimated by physical dosimetry (25 cGy). Correlation between the end points of the bioassays and estimated physical dosimetry was low (r < 0.2); the only significant correlation found was for physical dose estimate and dates worked at Chernobyl (r = 0.4, P < 0.01), with those working soon after the accident receiving greater estimated doses.

Impact of age and environment on somatic mutation at the hprt gene of T lymphocytes in humans.

Analysis of two human populations for dependence of somatic mutation on age has revealed both similarities and differences. The studies performed employed peripheral blood lymphocytes and measured the efficiency with which these cells form clones in vitro (cloning efficiency, CE) and the frequency of cells with inactivating mutations of the hypoxanthine phosphoribosyltransferase gene (mutant frequency, MF). The people studied were between 19 and 64 years of age. In one population, composed of 78 never smokers and 140 current smokers from the United States (US), both CE and MF were dependent on age: CE declined with age (p = 0.005); MF increased 0.15 per 10(6) cells per year of age for nonsmokers (p < 0.001) and at 1.3 times that rate for smokers (p = 0.01). In the second population, 80 people of unknown smoking status from Russia, the increase in MF per year was even greater, 2.5 times that of the US nonsmokers (p = 0.001) but the dependence of CE on age was the same as for the US population (p = 0.043). Because the increase of MF of the Russians with age is 2-fold greater than that of the US smokers, the intensity of smoking and/or other environmental exposures, or the susceptibility to these exposures, must account for the difference in age dependent MF increase, not the proportion of Russians that are smokers. Differences in the lymphocyte subpopulations that survived the longer transit from Russia may have contributed to the observed differences in MF. However, overall, the mutant frequency results suggest that the Russians were chronically exposed to higher levels of agents that induce somatic mutation and that, on an age adjusted basis, the Russia population studied is at increased risk for health consequences from accumulated genetic damage.

Elevated frequency of glycophorin A mutations in erythrocytes from Chernobyl accident victims.

In 1986, when an explosion accident occurred at the Chernobyl, Ukraine nuclear power plant, a large number of people were exposed to significant amounts of ionizing radiation. During the time between 1986 and 1992, peripheral blood samples were obtained from 102 people who either were on site during the emergency or were brought to Chernobyl shortly thereafter to assist in the cleanup of radioactive contaminants and isolate the damaged reactor from the environment. These blood samples plus samples from 13 unexposed Soviet individuals were analyzed by flow cytometry using the allele-loss somatic mutation assay for glycophorin A. Results of these assays show that the frequency of N/O variant red cells increased in proportion to the estimated radiation exposure of each individual. The radiation dose-response function derived from this population closely resembles that determined previously for atomic bomb survivors whose blood samples were obtained and analyzed 40 years after their exposure. This suggests comparable mutation induction per unit dose for these two populations and long-term persistence of the mutational damage. In addition, measurements on multiple blood samples from each of 10 donors taken over a 7-year period showed no significant changes in N/O variant cell frequencies, confirming the persistence of radiation-induced somatic mutations in long-lived bone marrow stem cells.

[Single cycles of direct and reverse enzymatic reaction illustrated by malate dehydrogenase]. / Edinichnye tsikly priamoi i obratnoi fermentativnoi reaktsii na primere malatdegidrogenazy.

Changes in the duration of self tryptophane fluorescence of soluble cytoplasmic malate dehydrogenase (MDG) and kinetics of chemical transformation in the course of single cycles of direct and reversible reaction L-malate in equilibrium with oxalacetate were recorded. MDG was shown to catalyse the direct and reversible reactions at different conformational states. The reaction coordinates for direct and reversible processes do not coincide in this case.

Parvalbumin conformers revealed by steady-state and time-resolved fluorescence spectroscopy.

The binding of calcium to whiting (one tryptophan residue) and pike (one tyrosine residue) parvalbumins has been studied by means of kinetic and steady-state fluorescence techniques. The decay curves of the tryptophan and tyrosine fluorescence of the parvalbumins are best fitted by a sum of two exponents for any metal state of the proteins. The data can be interpreted as a nonexponential decay of the fluorescence of a single-type chromophore or in terms of equilibria between compact and relaxed conformers of the parvalbumins in each metal state. Fluorescence quenching by I-ions and effects of H2O/D2O substitution confirm the second interpretation. The constants of the equilibria have been evaluated.