U R T S FI: FISH Biological Dosimetry - NTP - SOFT BACK 14-03-00 11:15:40 Rev 15.01 fi$$$$006p 45 Radiation Protection Dosimetry Vol. 88, No. 1, pp. 45–49 (2000) Nuclear Technology Publishing EXPERIENCE WITH FISH-DETECTED TRANSLOCATIONS AS AN INDICATOR IN RETROSPECTIVE DOSE RECONSTRUCTIONS S. Pressl†, H. Romm†, B. B. Ganguly‡ and G. Stephan† †Institute of Radiation Hygiene of the Federal Office for Radiation Protection Ingolstaedter Landstrasse 1, D-85764 Oberschleissheim, Germany ‡Cell Biology Division, BHABHA Atomic Research Centre Mumbai-400085, India Abstract — The prerequisite for the use of translocations as an indicator in retrospective dose reconstructions, is knowledge of the background level, persistence, and the availability of dose response curves for the conversion of translocation frequencies into doses. The results obtained in these areas are summarised. Cells with complete painted chromosome material are evaluated. Those showing any aberrations which involve painted material are stored in a computerised system, and described in detail. The simultaneous painting of whole chromosomes and centromeres has proved to provide a better level of discrimination between translocations and dicentrics. Following irradiation, direct proportionality was observed between DNA content covered by the painted chromosomes (11–19%) and the translocation frequency. The background level of translocations was determined in 42 healthy subjects, aged between 21 and 73 years of age. The statistical analyses of the data revealed no influence from sex and smoking habits on the translocation frequency. A clear increase in translocation yield was, however, observed for age. For the whole genome the frequency is at a level of 3 to 11 per 1000 cells, for all types of translocations. In a radiation accident victim (Estonia) the frequency of translocations was determined over a post-exposure time of four years. For two-way translocations, the half-life time was calculated to be 7.0 years, and that for one-way translocations 5.2 years. On the basis of our control data and our dose response curve, the lowest detectable radiation dose is about 0.3 Gy in subjects under 40 years of age, and about 0.5 Gy for those older than 40 years of age. INTRODUCTION Chromosome aberrations in cultured blood lympho- cytes have been shown to be reliable and sensitive indi- cators in the estimation of the dose to individuals and to population groups. Dicentrics represent here the pre- ferred aberration, since they are easily recognised and show a low background frequency in the general popu- lation. Dicentrics, however, are unstable with time and have limited value in estimating dose in people exposed over a long period of time or for assessments of exposures received in the distant past. Their analogues, translocations, persist with time, but with conventional staining methods such as banding, it has been difficult to analyse large numbers of them. Fluorescence in situ hybridisation (FISH) with whole chromosome-specific DNA probes can be used as a method for the rapid and precise analysis of translocations (1) . However, the prac- tical application of this method must be validated. In the following, our results, obtained by means of the FISH technique, concerning background level and persistence of translocations, are summarised. SUBJECTS, MATERIAL AND METHODS Subjects In order to study the background level of symmetrical translocations, peripheral blood samples were taken from 42 healthy subjects who were between 21 and 73 years of age. Of them 24 were men and 18 were women. Demographic data, information concerning smoking and drinking habits, exposure to ionising radiation, intake of drugs and recent infections, were obtained by interview and filling in a questionnaire. With regard to smoking habits, subjects were subdivided into the following groups: non-smokers who had never smoked, smokers with a daily consumption of 20 cigarettes, those who consumed 30 cigarettes per day, and ex-smokers who had stopped smoking 3 years before blood sampling. In order to investigate the influence of painted chro- mosomes covering different DNA contents on translo- cation frequency a blood sample was taken from a 22 year old woman. Following irradiation with 1 Gy of X rays, a culture was initiated and slides were prepared. As a consequence, blood sample, irradiation and culture conditions were identical for all slides. Finally, the fol- lowing chromosomes were painted in combination: 2, 4 and 8; 4, 13 and 22; 8, 15 and 16. The DNA content covered varies in accordance with the combination used, to a level of between 11.1 and 19.0 % (Table 1). Lymphocyte culture and chromosome preparation The standard protocol was used for lymphocyte cul- ture and chromosome preparation: 0.5 ml of freshly drawn venous blood was added to 4.5 ml RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mM glutamine, 2% PHA and antibiotics. The cultures were incubated at 37°C and treated with 0.1 g.ml -1 colce- mid for the final 3 h of the total culture time of 44 h.