The decrease of unstable chromosomal damages (dicentrics, rings and acentric fragments) was observed in circulating peripheral blood lymphocytes after exposure to ionizing radiation. The study comprised 100 subjects, all medical staff occupationally exposed to X-rays, who were re-examined for unstable chromosomal aberrations after 3 months (12 subjects), 6 months (31 subjects), 9 months (13 subjects), 12 months (21 subjects), over 24 months (14 subjects), and over 36 months (9 subjects) and 60 control subjects who were not exposed to ionizing radiation or chemical mutagens at their workplaces. The results did not show time-dependent recovery from chromosomal aberrations in all cases.Pad broja nestabilnih oštećenja kromosoma (dicentrični kromosomi, prstenasti kromosomi i acentrični fragmenti) praćen dulje vrijeme u limfocitima periferne krvi osoba koje su bile izložene ionizirajućem zračenju upozorio je na to da limfociti mogu preživjeti više godina nakon ekspozicije. Ovim istraživanjem obuhvaćeno je 100 osoba profesionalno izloženih djelovanju X-zraka. Praćene su nestabilne kromosomske aberacije u vremenskim intervalima 3 mjeseca (12 osoba), 6 mjeseci (31 osoba), 9 mjeseci (13 osoba), 12 mjeseci (21 osoba), 24 mjeseca (14 osoba), 36 mjeseci (9 osoba) i 60 osoba koje nisu bile izložene djelovanju ionizirajućeg zračenja ili kemijskim mutagenima na svojim radnim mjestima.
Dobiveni rezultati nisu pokazali popravak oštećenja ovisan o vremenu u svim slučajevima, što upućuje na individualne razlike u osjetljivosti na zračenje, kao i na različitu sposobnost za popravak oštećenih molekula

Karmela Šentija

Ružica Rozgaj

Vilena Kašuba

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1Arh hig rada toksikol 1998;49:1–8ORIGINAL SCIENTIFIC PAPERCHROMOSOMALABERRATIONS INMEDICAL STAFFOCCUPATIONALLYEXPOSED TO X-RAYS: AFOLLOW-UP STUDYVilena KA[UBA1, Ru`ica ROZGAJ1 andKarmela [ENTIJA2Institute for Medical Research andOccupational Health, Zagreb1,Clinical Hospital Merkur, Zagreb2,CroatiaReceived 24 October 1997The decrease of unstable chromosomal damages(dicentrics, rings and acentric fragments) wasobserved in circulating peripheral bloodlymphocytes after exposure to ionizing radiation.The study comprised 100 subjects, all medicalstaff occupationally exposed to X-rays, who werere-examined for unstable chromosomalaberrations after 3 months (12 subjects), 6months (31 subjects), 9 months (13 subjects), 12months (21 subjects), over 24 months (14subjects), and over 36 months (9 subjects)controls were 60 subjects who were not exposedto ionizing radiation or chemical mutagens at theirworkplaces. The results did not show time-dependent recovery from chromosomalaberrations in all cases.Key words:chromosomal damages, ionizing irradiation, occupationalexposureThe harmful effects of ionizing radiation were first recognized in occupationally ex-posed workers. Chromosomal aberrations are considered to be a sensitive biologicalindicator of the effects of ionizing radiation. They are a manifestation of DNA dam-age. They can also indicate alterations in the cell homeostasis which cause genomeinstability (1). Chromosomal abnormality (i.e. dicentric chromosomes) is formed soonafter irradiation and before the DNA replication (2). It is well known that X-rays pro-duce double strand breaks in DNA molecule (1). Strands rejoin rapidly while mostbreaks are repaired accurately (3–5). A small number of breaks remains unrepairedand becomes visible in metaphase chromosomes. In vivo analyses of chromosomalaberrations in peripheral blood lymphocytes in persons occupationally exposed toionizing radiation have shown various aberration frequencies. It has been well docu-mented that some lymphocytes containing aberrations continue to exist in the periph-eral circulation for many years after exposure (6, 7). In this study, we followed therecovery of DNA damage manifested as chromosome breakage in human peripheralblood lymphocytes in medical staff occupationally exposed to X-rays who were re-exami-ned at various intervals.2 Ka{uba V, Rozgaj R, [entija K: X-RAY INDUCED CHROMOSOMAL ABERRATIONS IN MEDICAL STAFFArh hig rada toksikol, Vol 49 (1998) No 1, pp. 1–8SUBJECTS, MATERIALS, AND METHODSWe examined 100 subjects for structural chromosomal aberrations (one or moredicentric and/or ring chromosome), all of them medical staff occupationally exposedto X-ray radiation. The examinees were divided into six groups classified according tothe period of re-examination, ranging from 3 to 36 months. A questionnaire designedto obtain relevant details with health history, current health status, lifestyle, and expo-sure to ionizing radiation was completed for each subject. None of the subjects hadpreviously been exposed to chemical mutagens. The control group of 60 subjectswas comparable in age and smoking habit. After the first examination of the exposedsubjects, analyses for chromosomal aberrations were repeated at intervals of 3 months(Group 1, N=12), 6 months (Group 2, N=31), 9 months (Group 3, N=13), 12 months(Group 4, N=21), < 24 months (Group 5, N=14), and < 36 months (Group 6, N=9).The presence of chromosomal aberrations was examined in the lymphocytesfrom whole peripheral blood. We used a standard 48-hour culture technique; the cellswere cultivated in F-10 medium (Gibco, GB), supplemented with PHA (PHA-Murex,GB), 20% fetal calf serum (Biological Industries, Israel) and antibiotics (penicillin–10000 IU/ml and streptomycin–10000 µg/ml). The slides were stained by Giemsasolution. The analysis of at least one hundred first metaphases per subject (100–200cells) was limited to structural aberrations such as chromatid and chromosome breaks,dicentrics, rings, chromatid exchanges (tetraradial) and acentric fragments.Intragroup differences in the number of chromosomal aberrations (acentric frag-ments, dicentric and ring chromosomes) between the first and second sampling pe-riods were evaluated by c2-test at the level of significance P<0.05.RESULTSThe results obtained at first and second examination (ranging in the period from 3 to36 months) are given in Figure 1 and Table 1. Table 2 summarizes all results of theexposed group. Table 3 shows the results of the control group.3Ka{uba V, Rozgaj R, [entija K: X-RAY INDUCED CHROMOSOMAL ABERRATIONS IN MEDICAL STAFFArh hig rada toksikol, Vol 49 (1998) No 1, pp. 1–8In comparison to the number of chromosomal aberrations observed on the firstexamination, the findings for 25% of examinees remained unchanged after the re-exami-nation three months later, whereas 8.3% of subjects maintained an increase, and66.7% a decrease in chromosomal aberrations (Table 1 and 2).Figure 1 Percentage of unstable chromosomal aberrations in control subjects and in exposedsubjects at first and second sampling times: after 3 months (Group 1), 6 months (Group 2),9 months (Group 3), 12 months (Group 4), <24 months (Group 5) and <36 months (Group 6).     Control subjectsfirst samplingsecond samplingGroups of subjects% of unstable aberrationsExposed subjects4 Ka{uba V, Rozgaj R, [entija K: X-RAY INDUCED CHROMOSOMAL ABERRATIONS IN MEDICAL STAFFArh hig rada toksikol, Vol 49 (1998) No 1, pp. 1–8Table 1 Chromosomal aberration analysis in six groups of subjects occupationally exposed toX-raysChromatid Chromosome Acentric Dicentric Ring ChromatidGroupbreak break fragment interchangesN (tetraradial)% % % % % %First 1.35 0.83 2.23 0.93 0.04 0.041 12 sampling (0.5 – 3.8) (0.5 – 3.6) (0.5 – 11.3) (0.5 – 3.8) (0.5) (0.5)Re-examination 1.26 0.57 1.19 0.33 0.08 0.043 months later (0.5 – 2.5) (0.5 – 2.0) (0.5 – 2.5) (0.5 – 1.0) (1.0)     (0.5)First 0.93 1.5 2.39 1.05 0.20 0.032 31 sampling (0.5 – 3.0) (0.5 – 4.8) (0.5 – 13.0) (0.5 – 5.5)(0.5 – 1.0) (1.0)Re-examination 0.83 1.33 1.51 0.43 1.19 0.026 months later (0.5 – 4.0) (0.5 – 3.8) (0.5 – 4.2) (0.5 – 2.5)(0.5 – 1.8) (0.1 – 0.5)First 1.05 1.03 3.37 1.27 – 1.153 13 sampling (0.5 – 4.0) (0.5 – 2.5) (1.0 – 16.0) (0.5 – 3.3) – (0.5 – 1.4)Re-examination 0.64 2.35 1.09 0.19 – 0.089 months later (0.5 – 1.5) (0.5 – 9.0) (0.5 – 2.0) (1.0 – 1.5) – (0.5)First 0.86 2.74 2.69 1.49 0.09 0.084 21 sampling (0.5 – 2.0) (0.5 – 9.0) (0.5 – 11.9) (0.5 – 5.5)(0.5 – 1.3) (0.6 – 1.0)Re-examination 1.32 1.64 0.99 0.3 0.02 0.0212 months later (0.5 – 3.0) (0.5 – 5.0) (0.5 – 4.5) (0.5 – 2.0) (0.5) (0.5)First 1.29 2.04 1.69 0.94 0.04 0.065 14 sampling (0.7 – 3.5) (1.6 – 5.0) (1.0 – 7.2) (0.5 – 1.8) (0.5) (0.8)Re-examination 1.39 1.45 1.03 0.27 0.13 –< 24 months later (0.7 – 6.3) (0.5 – 4.5) (0.6 – 2.0) (0.5 – 1.1) (1.8) –First 1.73 1.19 1.41 0.79 0.24 0.16 9 sampling (1.0 – 2.0) (1.0 – 3.3) (1.0 – 3.0) (0.7 – 1.5)(0.9 – 1.3) (1.0)Re-examination 1.51 1.24 1.42 0.54 0.2 0.1< 36 months later (0.5 – 3.5) (0.5 – 3.0) (0.5 – 3.5) (0.5 – 1.9) (1.8) (1.0)Results are presented as percentages (range in brackets).5Ka{uba V, Rozgaj R, [entija K: X-RAY INDUCED CHROMOSOMAL ABERRATIONS IN MEDICAL STAFFArh hig rada toksikol, Vol 49 (1998) No 1, pp. 1–8Table 2 Number of changed findings of chromosomal aberrations in the exposed subjectsGroup N Increase Decrease Unchanged1 12 1 8 3(8.3) (66.7) (25)2 31 8 22 2(25) (68.8) (6.2)3 13 0 12 1(92.3) (7.7)4 21 2 17 2(9.5) (81) (9.5)5 14 1 8 5(7.1) (57.2) (35.7)6 9 2 6 1(22.2) (66.7) (11.1)Results are presented as absolute numbers (percentage in brackets).Table 3 Chromosomal aberration analysis in the control subjectsAge No. of No. of Chromatid Chromosome Acentric Dicentric Chromatid(range) persons cells break break fragment interchangesSex (tetraradial)% % % % %Men 25–36 32 4036 0.5 0.8 0.4 0.05 –Women 19–46 28 4679 0.7 1.3 0.4 – 0.02Total 19–46 60 8715 0.6 1.0 0.4 0.02 0.01Regardless of the results, all examined subjects, except nine, have continued towork. As indicated by film dosimeter, three subjects received the exposure dose thatexceeded currently recommended dose limits (the current whole-body dose limit inCroatia is 50 mSv for persons occupationally exposed to radiation).We also observed multiply damaged cells containing more than one dicentric orring, even some tricentrics, and nearly 20 acentric fragments.6 Ka{uba V, Rozgaj R, [entija K: X-RAY INDUCED CHROMOSOMAL ABERRATIONS IN MEDICAL STAFFArh hig rada toksikol, Vol 49 (1998) No 1, pp. 1–8DISCUSSIONThe estimation of chromosomal aberrations in human peripheral blood lymphocytesis an important technique for risk assessment in occupational exposure (8). Most cellsrepair radiation induced damages shortly after the occurrence (5, 9). However, somebreaks remain unrepaired or repair wrongly. These changes may be observed aschromosomal aberrations in metaphase cells.Figure 2 shows the chromosomes from a single lymphocyte taken from a radi-ologist who worked for 12 years in a radiation zone. It displays a selection of types ofaberrations encountered in cytogenetic analyses. All these aberrations are termedunstable because they hinder the normal separation of genetic material during celldivision. These aberrations are likely to be lethal within a few cell cycles and would beselectively eliminated from a naturally proliferating population of cells.Figure 2 A metaphase spread from a human lymphocyte exhibiting unstable chromosomalaberrations. b – dicentric, t – tricentric, a – acentric fragment, m – minute, g – gap. (Not all theaberrations have been labeled in this figure).It is well established that cells containing aberrations slowly disappear from thecirculating lymphocyte pool (10, 11). The results of our study show that the recoveryfrom chromosomal aberrations observed at various intervals of blood sampling is notsignificantly time-dependent. Summarizing all results, Table 2 shows a decrease ofchromosomal aberrations in 92.3% and 81% of subjects reexamined after 9 and 12months respectively. The number of dicentrics or ring chromosomes was not alwaysfollowed by a corresponding number of acentric fragments.We analyzed and compared individual differences in the number of chromosom-al aberrations within each group and between the groups. Individual differences in7Ka{uba V, Rozgaj R, [entija K: X-RAY INDUCED CHROMOSOMAL ABERRATIONS IN MEDICAL STAFFArh hig rada toksikol, Vol 49 (1998) No 1, pp. 1–8radiosensitivity, as well as different repair ability were also considered. The cell has anumber of mechanisms to repair DNA damage. Rapid and flawless repair systemscan compensate for much higher irradiation doses than the slow and error-proneones can. Oesch and co-workers (12) showed that the same individual has a differentrepair capacity at different times, which is a consequence of differences in endoge-nous physiological status or changing exposure to exogenous compounds. It is alsoknown that human lymphocytes consist of cell sub-populations with different radio-sensitivity (13). There is evidence to suggest differences in sensitivity as a function ofcell-cycle position. Using the comet assay in different mammalian cell lines, Oliveand Banath showed that damage repair also depended on cell-cycle position (4).After a partial or random exposure, cells from non-irradiated areas or areas irra-diated with low doses might reduce the average aberration frequency (14). It is there-fore difficult to define the period for which the unstable aberration frequency observedin lymphocytes can be used directly for dosimetry.The results point out the importance of chromosomal aberration analysis as ameans of the detection of radiation induced damage, as well as the significance ofimplementation of protective measures. It is also important to stress the necessity ofpre-employment analysis for every person who is to start to work in an ionizing radi-ation zone. The individual differences in radiosensitivity as well as the frequency ofcytogenetic aberrations depend on physical condition of each subject and on thevariability in DNA repair processes. As time-dependent changes in dicentrics may besubject to individual variations, it may be difficult to extrapolate from one case toanother (14). The present results do not allow definitive conclusions about low-doseionizing radiation during the occupational exposure, but are intriguing enough tomotivate further investigation.REFERENCES1. Galloway SM. Chromosome aberrations induced in vitro: mechanisms, delayed expression,and intriguing questions. Environ Mol Mutag 1994;23(Suppl 24):44–53.2. Natarajan AT. Mechanisms for induction of mutagenesis and chromosome aberrations. Envi-ron Health Persp 1993;101(Suppl. 3):227–31.3. Borek C. Molecular mechanisms in cancer induction and prevention. Environ Health Persp1993;101(Suppl. 3):237–45.4. Olive PL, Banath JP. Induction and rejoining of radiation-induced DNA single-strand breaks:»tail moment« as a function of position in the cell cycle. Mutation Res DNA Repair 1993;294:275–83.5. Fortini P, Raspaglio G, Falchi M, Dogliotti E. Analysis of DNA alkylation damage and repairin mammalian cells by the comet assay. Mutagenesis 1996;11:169–75.6. Awa AA. Review of thirty years study of Hiroshima and Nagasaki atomic bomb survivors. II.Biological effects. Chromosomal aberrations in Somatic Cells. J Radiat Res 1975;16(Suppl):122–31.7. Leonard A, De Knudt Gh, Leonard ED. Persistence of chromosomal aberrations in an acciden-tally irradiated subject. Radiat Protect Dosim 1988;22:55–7.8. Bauchinger M. Cytogenetic effects in human lymphocytes as a dosimetry system. In: EisertWG, Mendelsohn ML, eds. Biological Dosimetry. Berlin: Springer. 1984:15–24.8 Ka{uba V, Rozgaj R, [entija K: X-RAY INDUCED CHROMOSOMAL ABERRATIONS IN MEDICAL STAFFArh hig rada toksikol, Vol 49 (1998) No 1, pp. 1–89. Olive Pl, Banath JP, Durand R. Heterogeneity in radiation-induced DNA damage and repair intumor and normal cells measured using the »comet« assay. Radiat Res 1990;122:86–94.10. Guedeney G, Grunwald D, Malabert JL, Doloy MT. Time dependence of chromosomal aber-rations induced in human and monkey lymphocytes by acute and fractionated exposure to60Co. Radiat Res 1988;116:254–62.11. Ramalho AT, Curado MP, Natarajan AT. Lifespan of human lymphocytes estimated during asix year cytogenetic follow-up of individuals accidentally exposed in the 1987 radiological ac-cident in Brazil. Mutat Res 1995;331:47–54.12. Oesch F, Aulmann W, Platt KL, Doerjer G. Individual differences in DNA repair capacities inman. Arch Toxicol 1987;(suppl 10):172–9.13. Wuttke K, Streffer C, Muller W-U. Radiation induced micronuclei in subpopulations of humanlymphocytes. Mutat Res 1993;286:181–8.14. Doloy MT, Malabert JL, Guedeney G, et al. Use of unstable chromosomal aberrations forbiological dosimetry after the first postirradiation mitosis. Radiat Res 1991;125:141–51.Sa‘etakPRA]ENJE KROMOSOMSKIH ABERACIJA U MEDICINSKOG OSOBLJAPROFESIONALNO IZLO@ENOG DJELOVANJU RENDGENSKIH ZRAKAPad broja nestabilnih o{te}enja kromosoma (dicentri~ni kromosomi, prstenasti kromosomi i acentri~ni fragmenti)pra}en dulje vrijeme u limfocitima periferne krvi osoba koje su bile izlo‘ene ioniziraju}em zra~enju upozorio je nato da limfociti mogu pre‘ivjeti vi{e godina nakon izlo`enosti. Ovim istra‘ivanjem obuhva}eno je 100 osobaprofesionalno izlo‘enih djelovanju rendgenskih zraka. Pra}ene su nestabilne kromosomske aberacije u izlo`enimosobama u vremenskim intervalima 3 mjeseca (12 osoba), 6 mjeseci (31 osoba), 9 mjeseci (13 osoba), 12 mjeseci(21 osoba), <24 mjeseca (14 osoba), <36 mjeseci (9 osoba) i u 60 osoba koje nisu bile izlo‘ene djelovanjuioniziraju}eg zra~enja ili kemijskim mutagenima na svojim radnim mjestima.Dobiveni rezultati nisu pokazali popravak o{te}enja ovisan o vremenu u svim slu~ajevima, {to upu}uje naindividualne razlike u osjetljivosti na zra~enje, kao i na razli~itu sposobnost za popravak o{te}enih molekula.Klju~ne rije~i:ioniziraju}e zra~enje, o{te}enja kromosoma, profesionalna izlo‘enostRequests for reprints:Vilena Ka{uba, Ph.D.Institute for Medical Research and Occupational Health2 Ksaverska St., P.O. Box 291,HR–10001 Zagreb, CroatiaE-mail: vkasuba@mimi.imi.hr

Praćenje kromosomskih aberacija u medicinskog osoblja profesionalno izloženog djelovanju rendgenskih zrka

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