Radio! Oncol 19%; .30: 297-304. Incidence of spontaneous cytogenetic changes in peripheral blood lymphocytes of a human population sample Marjan Bilban, Sonja Vrhovec Republic of .Slovenia Inslilute of Occupational Safety, Ljttb/jaiia, Slovenia Three nnilagene/ic me//iocls - conven/idnal s/ruc/iira/ chromosomal aber/a/ion ana/vsis (CA), sisley chromatid exchange (SCE) me/hod and micronucleus /es/ ( MN) were carried ««t «/i a popii/a/ion sample o/ .350 /es/ siib/ec/s agecl /8' /o 65 years. 7'he spoii/wieoiis incidence o/ /hese aberra/ions in /he /irs/ in vi/ro me/aphases of'lyvnphocvtes was 0.8. % ( hroma/icl breaks, 0.4. % isochronw/id-chromosome. breaks, 0.2/ % acen/rics /ragmen/s and O.OJ % i/icen/ric i hro/noso/iies per /es/ sub/ec/. 7'he mean va/i/e /or SCE per cel/ a/noim/ed t« 6.52 ± 0. 70, while the frequency of /iiicronuclei was 5.8 ± 2. I per 500 hi/iiic'lear /vmphocv/es. These resii//s represent /he iinilagenelic background for /he Slovenian /•opit/a/ion a/ic/ can be i/sedfor !he assessment or i/i case ofsuspecl expos/ire /o clas/ogenic agen/s. Key words: Lymphocytcs: chromosomc aberrations: sister chromatid cxchangc; microimclci Introduction Intensive industrialisation over the past few decides has resulted in the production and use of numerous genotoxic chemicals and sources of ionising and non-ionising radiation. The need to identify the mutagenic and carcinogenic effects of these agents on the human population exposed environmentally. professionally or accidentally is therefore on the increase. There are severa! methods with which it is possible to prove changes occurring in DNA molecules. Unfortunately, there are few direct methods for the identification and assessment of the degree of mutations. Among methods for the detection of large chang-cs in the genome of human somatic cells which are used routinely for mutagenetic monitoring, the ln-ternational Commission for Protection against Environmental Mutagens and Carcinogens recom- Correspondence to: Marjan Bilhan. Assist. Dr. Sc.. MD. Republic of Slovenia Institute of Occupational Safety. Bohoričeva 22a. Ljubljana. Slovenia. UDC: 6K).I55.32-09I.85:6I6.I 12.9-1 4.24 mends thc detection of chromosomal aberrations, the micronucleus test and sister chromatid exchangc technique.1 Thc analysis of chromosomal aberrations in peripheral blood lymphocytes has gained the widest use to date. The methodological and technical conditions for this technique and the procedure lor the analysis of specimens are well defined, largely owing to the use of specific chromosomal aberrations in biological dosimetry.2 It is well known, however, that ionising radiation and the majority of chemical genotoxic agents have different effects on cellular DNA which is directly included in the formation of chromosomal changes. The frequency of SCE is a sensitive indicator of the effects of chemical genotoxic agents and high linear energy transfer (LET) radiation, but a poor indicator of the exposure to low LET ionising radiation. u The micronucleus test has almost universal application in the detection of changes in the cellular genome. Micronuclei may originate from acentric fragments resulting from two-chain breaks of DNA alter its exposure to radiation, and have shown very 2')8 Bilbim M and Vrhovec S g,)od dose-response relationships. They may also contain several chromosomes which were not distributed equally to daughter cells due to a nonfunctional cell-division spindle or kinetochores. The hitter phenomenon is most frequently caused by chemical agents.5 It therefore seems that all three tests should be used on parallel specimens in order to be able to assess the type of exposure: to a physical or chemical agent, or even possibly simul-t; ■ neously to both categories of clastogens.'' To enable the evaluation and correct performance of cytogenetic population monitoring, it is necessary to know as much as possible about the liequency of normal, spontaneous chromosomal aberrations. as well as about the factors which exert au influence on their occurrence.7 Considering the institutions in which the authors of this paper work and the legal obligation of mutagenetic monitoring for defined groups of people who are professionally exposed to ionising radiation, the purpose of this study was: - to standardise laboratory conditions of in vitro cultivation of lymphocytes in accordance with the Protocol of the International Atomic Energy Agency, - to determine the mutagenetic background in a population sample of persons who are not professionally exposed to ionising radiation by using the siructural chromosomal aberration analysis, micro-nucleus test and SCE frequency per cell. Subjects and methods Subjects The study included 350 subjects, of whom 153 were s1 udents prior to their enrolment at the High School of Radiology, and 197 persons prior to the assumption of their duties in radiation zones. It is important to emphasise that such a selection of the population sample enables comparative analyses regarding mutagenic effects in conjunction with age, and even more, lifestyle factors. The group of students included secondary school s udents who had just graduated, aged 18 to 20 years. The majority of them were non-smokers and persons with an extremely low previous influence of alcohol, coffee, drugs and ionising radiation used for diagnostic and treatment purposes. The second group of test subjects had not been professionally exposed to genotoxic substances before taking of blood samples for mutagenetic tests. However, the age dispersion in this group (20 to 65 years) was significant, and the lifestyles of its subjects had undoubtedly brought along more numerous factors with mutagenic characteristics. At the time when blood samples for mutagenetic tests were taken, none of the test subjects showed any subjective troubles nor objective signs of acute illness, and none of them had been previously subject to major diagnostic or therapeutic irradiation. Test subjects were both men and women, of whom 34 % were smokers. Methods Data of subjects was collected by means of filling in Personal Health Questionnaires. Peripheral blood lymphocytes from subjects were used as cellular material. Blood samples were taken simultaneously for all three tests. Structural chromosomal aberrations (CA) Standard in vilro lymphocyte cultures were used for structural chromosomal aberration analysis. 0.3 ml of heparinised whole blood was addecl to 5 ml of the culture medium (GIBCO BRL Chromosome med I A with Phytohaemaglutinin). The first in vitro cell division cycle was established with an addition of 5 mg/ml of BrclU (Sigma). 0.075 mol/1 potassium chloride was used for the hypotonic procedure. Fixation was performed in a mixture of ice acetic acid and methyl alcohol at a ratio of 1:3. The cell suspension was pipetted onto cold glass slides, specimens were air-dried and stained with Giemsa-Sigma. For every test subject, the first 200 in vilro metaphases were analysed at IOOOX magnification on a Nikon LABO-PI-IOT2 microscope. Structural damage to chromosomes was categorised as chromatid breaks, isochromaticl-chromosomal breaks, acentric fragments or dicentric and ring chromosomes. Gaps were not included in the total number of chromosomal aberrations. Sister chromatid exchange (SCE) The same culture medium was used as for the first test. 72-hour lymphocyte culiures with the addition of IO mg/ml BrdU were prepared in dark conditions. The procedure was performed according to the Kato (1974) method.0 //«■/V/ern'e o/',vpo///ii//eoH.v cy/ogciicric in peripheru/ Wood /yM/phorv/e, 1,111111111 population sttmp/e 299 50 cells per subject were analysed, SCE were counted and presented as average numbers per celi. Tile range of SCE frequencies was also recorded lor every subject. Micronucleus test {MN) For this test, 3 mg/ml of cytochalasin B (Sigma) was added to each in vitro lymphocyte culture in the 43rd hour of cultivation. The Fenech-Morley ( 1985) method was used.'' Hypotonic procedure was omitted, and specimens were stained according to May-Griinwald and Giem-sa. Cells with clearly blocked cylokinesis (CB cells), i.c. binuclear cells, were analysed. 500 cells per pt-rson were inspcctcd and the resulls were presented as ihe number of rnicronuclei per 500 CB cells. Statistical dala processing Dala was processed using standard methods of parametric statistics. The differences between the average values for individual groups were tested using the variance analysis method with the SPSS computer program. Results Structural chromosomal aberrations (CA) were analyzed in 153 students or 30,600 their first in vitro metaphases, and in 197 technicians pending employment or 39400 their first in vitro metaphases (Table 1). The average value of acentric fragments for technicians pending employment is 0.58 and for students only 0.24. Acentric fragments were found Table l. Mutagenetic testing of subjects - Structural chromosomal aberrations % liberations Nn Tests All subjects _____ ............................. All subjects Subjects with acentrics Groups of test subjects No. of subjects No. of exam. cells Mean SD Mean SD Share of subj. (%) Mean SD o 1 2 3 4 5 6 7 8 9 10 1 2 .3 Technicians .Students Total 197 153 .350 .39400 30600 70000 1.812 1,101 1,501 1,028 0,580 0,861 0,584 0.242 0.434 1.045 0.473 0,860 39.090 22,880 32,000 1.494 1,057 1,357 1,199 0.338 1.029 Variance FR analysis FP 58.647 0,000 14,1 33 0.000 4.455 0,037 RINGS DICENTRIC CROMOSOMES All subjects Subjects with rings All subjects Subjects with dicentric chrom. Mean SD .Share ol' subj. (%) Mean SD Mean SD Share of subj. (%) Mean SD 11 12 13 14 15 16 17 18 19 20 0.005 0,000 0,003 0.071 0.000 0,054 0,508 0,()00 0,286 1.000 1,000 0,041 0.000 0,023 0.283 0,000 0,211 2,538 0,000 1.429 1,600 0.000 1,600 0,894 0.000 0,894 0,776 0,379 3,153 0,077 CHROMOSOMAL BREAKS CHROMATID BREAKS All subjects Subjects with chromosomal br. All subjects Subjects with chromatid br. Mean SD Share of subj. (%) Mean SD Mean SD Share of subj. (%) Mean SD 21 22 23 24 25 26 27 28 29 30 1,157 0.980 73.100 1,583 0,798 1,838 0,928 97.460 1,885 0,891 0,4'!7 0,660 41.180 1,206 0,446 1.464 0,726 93,460 1,566 0.634 0,871 0,856 59,143 1.469 0,729 1,675 0,860 95,714 1,749 0,806 1.418 12.358 16,801 13,313 0,000 0,001 0,000 0,000 300 ßi/bim M and Vrhovec S in 22.88 % of students (1.05 on average) and in 39.09 % of technician.s pending employment (1.49 011 average). In one case, the presence of a ring chromosome was found in a technician pendingem-ployment, while such aberrations were not found in siudents. The average value lor rings is therefore 0.00,5 I per subject for technicians. In five persons same population, dicentric chromosomes were i'i>und. The average value of dicentric chromosomes li>r this group is 1.6, and 0.0406 for all technicians. The number of chromosomal breaks in technicians is 1.15 per subject and only 0.49 in students: for chromatid breaks, it is 1.83 per subject and 1.46 iii students. Chromosomal breaks were present in 41.18 % of si udents (1.20 on average) and in 73.10% of technicians (1.58 on average): chromatid breaks were present in 93.46 % of students (1.56 on average) and in 97.46 % of technicians (1.88 on average). 1 he differences between the groups of students and technicians pending employment were found to be statistically significant for the percentage CA test, the number of acentric fragments, chromatid and chromosomal breaks. The SCE and MN tests were performed only on a smaller number of test subjects. The average result of the SCE test for students was 6.28 and 6.52 for technicians per 50 clls: for MN test the average results were 4.00 for siudents and 5.89 per 500 CB cells for technicians. The differences between the two compared groups were not statistically significant (Table 2). It can be seen from mutagenetic questionary that during the present length of service examined subjects had not worked with sources of ionising radiation. There is a considerable difference in the total duration of service between the two groups, since the technicians have in average up to 7 years of service, while the majority of students have none. The age differences are also significant, since the technicians are on average ii years older than students. With the comparison of mutagenetic tests for the two groups we have established that the percentages of CA, the number of acentrics, chromosomal and chromatid breaks increase almost linearly with age. The differences in age groups are statistically significant in % CA, acentrics and chromosomal breaks (Table 4). While examining the influence of smoking on our results it was established that there were no significant differences in mutagenetic tests between smokers and non-smokers, except for the values for leukocytes (which are higher than average both in technicians and in students who smoke). In these two groups, the SCE test was performed only on a small number of subjects. In their smoking history is very short (smoking history for students is 1.5 years). .Student smokers (26 %) smoke on average only 8 cigarettes per day, while for technician smokers (41 %) the average duration of smoking is 6.8 years and they smoke on average i4 cigarettes per day). 'J able 2. Mutagenetic testing of subject - SCE and MN test. Tests SCETEST MINTEST o. Groups ^^^^ of test suhj. No. of ^subjects No. of exain. cells Mean - per 50 cells SD No. of subjects No. of exam. cells Mean - per 500 CB cells SD ■) 1 2 3 4 5 6 7 8 9 Technicians 2 Students ? Total 115 5 120 5750 250 6000 6.528 6,268 6,517 0,714 0,292 0,702 113.000 5.000 118.000 56500,000 2500.000 59000.000 5,8% 4,000 5,815 2.122 1,581 2,132 Variance FR analysis FP 0,651 0,421 3,880 0,051 'lablc 3. Length of service and age of test subjects. Variable Length of service - at present (years) Lenth of service — total (years) Age (years) o. Groups ^^^^ No. of No. of No. of of icsi suhj. ^^^ subjects Mean SD subjects Mean SD subjects Mean SD -) 1 2 3 4 5 6 7 8 9 10 1 Technicians 197 0.000 0.000 197 6,944 8.587 197 32.906 9.395 > Students 153 0,059 0.367 153 0,059 0.367 153 21,597 1.983 S Total 350 0.026 0,244 350 3,917 7.279 350 27,934 9.092 Variance FR 5,066 98,182 214,057 analysis FP 0.025 0.000 0,000 h/nWeme ofsi>oiitniieoux cytogenetic c/iaiiges in peripheral /jlood /y///phon7es »/'« lunntni p«p»/r/fi»M .«//»p/e .30! Table 4. Mutagenetic tests according to agc of groups; of .subjects; - Structural chromosomal aherralions. ~~~-■—Tests "/"CA ACENTRICS No. Groups of test subj/ects ~---—All subjects All suhjccls .Subjects wilh acentric. Agc groups of No. of subjects (years) subjects No. of cxarn. cells Mean SD Mean SD Share of subj. (%) Mean SD (1 ! 2 3 4 .5 6 7 8 9 10 1 Till 18 5 1000 1.000 0.354 0,200 0.447 20.000 1,000 0,000 2 19-25 183 36600 1,195 0,666 0,236 0.487 21.311 1,103 0.384 ?. 26-35 102 20400 1.639 0,693 0.446 0.640 36,274 1.216 0.417 4 .36-45 39 7800 2,218 1.455 0.923 1.345 51,282 1.800 1.399 5 46-55 13 2600 2.269 1.666 1,308 2.394 61.538 2,125 2,800 (, 56-65 8 1600 2.438 0.729 1,250 0.707 87.500 1.429 0.535 Total 350 70000 1,504 0,849 0.437 0.812 32,000 1.357 1.030 Variance FR analysis FP 15,255 0,000 9.695 0,000 2.421 0,040 RINGS DICENTRIC CHROMOSOMES All subjects Subjects with rings All subjects Subjects with dicentric chrnrn. Mean 11 SD 12 Share of subj. (%) 13 Mean 14 SD 15 Mean 16 SD 17 Share of subj. (%) 18 Mean 19 SD 20 0,000 0.000 0.000 0.000 0.000 0,000 0.000 0,000 0,000 0.000 0,000 0.000 0,000 0,011 0,105 1,093 1,000 0.000 0.000 0.000 0,000 0.000 0.000 0.000 0.000 0.000 0,000 0,000 0.000 0.000 0.000 0.128 0.570 12.821 2.500 0.707 0,077 0.277 7.690 1,000 0.077 0,277 7,692 1,000 0.000 0,000 0,000 0,000 0.000 0.000 0,000 0,000 0,000 0.000 0.003 0.054 0,286 1.000 0,023 0,211 2.286 1,600 0.500 5.487 2.492 5.400 0.000 0,03! 0,156 CHROMOSOMAL BREAKS CHROMATID BREAKS All s ubjects Subjecls with chromosomal hr. All s ubjects Subjects with chromatid br. Mean SD Share of Mean SD Mean SD Share of Mean SD subj. (%) subj. (%) 21 22 23 24 25 26 27 28 29 30 0,600 0,548 60,000 1.000 0,000 1.200 0.447 100,000 1.200 0.447 0.571 0,745 44.262 1.300 0,560 1,571 0,803 94,536 1,663 0,727 1,099 0.878 72.549 1.500 0,667 1,733 0,747 97.059 1,786 0,692 1.43fi 1,071 87.179 1.647 0,981 1,949 1,297 97.436 2.000 1.273 1,231 0,927 76.923 1.600 0.699 1,846 0.801 100.000 1,846 0,801 1.625 1,598 62.500 2.600 1,140 2.000 1,069 87,500 2,286 0.756 0,871 0.856 59,143 1.469 0.729 1,675 0,860 95,714 1.749 0,806 1 1,091 4.322 2,047 2,333 0,000 0.001 0,072 0,042 Discussion The determination of the frequency of chromosomal aberrations in peripheral blood lymphocytes as a biological indicator of the effect of genotoxic agents h is been used in practice for a long period of time. During this time, a large number of papers were published which presented data on Ihe frequency of spontaneous aberrations. especially Ihose of the chromosomal type. With regard to the fact lhat the spontaneous incidence of genome damage is essen-lially influenced by individual non-idenlified agents and regional, ecologically specific features, we are interested in comparing the recent situation with values l'rom I 0 or more years ago. The most systematic analysis of this type was performed by Lloyd in 1984. Even though he focused only on the spontaneous occurrence of dicentric and acentric fragments, he also analysed other available data on chromatid and chromosomal lesions. 1 " He analysed 302 Bi/ban M and Vrhovec S Table 5. Mutagenetic te.sting ofsubjects -SCE and MN te.st.s. Tbsts SCETEST MNTEST No.Age No.of No. of Mean -per gp No. of No. of Mean -per groups ^~^^subjects exam. cell.s .50 cells ' subjects exam. cells 500CB cells ' of subj. (years) o 1 2 3 4 5 6 7 8 9 1 19-25 23 1150 6,228 0,658 23 11500 4.913 2.151 2 26-35 62 3100 6,466 0,551 61 30500 5,817 1,873 3 36-45 23 1150 6,737 1,021 22 11000 6,509 2,516 4 46-55 7 350 6,763 0.434 7 3500 6,286 1,976 5 56-65 5 250 7.096 0.664 5 2500 6,600 2.702 6 Total ■ 120 6000 6.517 0.702 118 59000 5,815 2,132 Variance FR analysis FP 2.816 0,029 1,921 0,112 Table 6. Length of service and age of test subjects according to age groups. No. Length of service - at present (years) Length ofservice - total (years) Age (years) No. of subjects No. ofexam. cells SD No. of subjects No. of exam. cells SD No. of subjects No. of exam. cells SD 0 1 2 3 4 5 6 7 8 9 10 I do 18 5 0.000 0,000 5 0,000 0,000 5 18,367 0,070 2 19-25 183 0.044 0,326 183 0,412 1,142 183 21,885 1,760 3 26-35 102 0.010 0,100 102 4,525 4,711 102 29,390 2,635 4 36-45 39 0.000 0,000 39 11,811 8,366 39 39,537 2,720 5 46-55 13 0.000 0,000 13 16,615 14,245 13 49,730 3,033 6 56-65 8 0.000 0,000 8 22,000 15.062 8 59,913 3.168 7 Total 350 0.026 0,244 350 3,917 7.279 350 27.934 9.092 Variance FR analysis FP 0.427 0.830 70,792 0,000 1074,692 0,000 a total of 65 different mutagenetic studies with over 2000 test subjects, i.e. 211,660 examined cells. The values for dicentric fragments are mainly in the range from I to (more rarely) 2/1000. Since his studies included test subjects from a wide age interval, our data differ somewhat from his. Not one dicentric fragment or ring chromosome was found in our group of students, while in the group of technicians (which was composed of subjects of greater age differences and longer smoking periods, as well as the influence of other lifestyle factors), dicentric and centric ring chromosomes were found in O.OI 1 %. Acentric fragments were found in both groups in 0.217 %. Lloyd stated that in the majority of publications he analysed, the value for acentric fragments was about 3 x 10-'. In a study which included .304 subjects, Galloway et al. (1986) found a frequency of dicentrics of 2.1 x 10-' and 3.2 x 10-' for acentric fragments.11 Bender et al. (1988) found 1.6 x 10-' dicentrics and 4.6 x 10-' acentric fragments in a mixed black-white American popula-tion.7 In addition to individual standard aberrations, Awa and Neei (1986) also stale data on the pres- ence of a certain number of "rogue" cells with multiple dicentric, tricentric and acentric fragments and breaks. No explanation for this phenomenon is given.1 2 Such types of changes were not found in this study, even though they were present in certain other of our studies. The comparative analysis of the frequency of chromatid aberrations revealed certain problems. This type of aberrations involves changes which usually do not originate from the circulating Go population of lymphocytes, but are formed either during or after the phase of DNA synlhesis, i.e. in v/'ira. There are also differences regarding the classification of chromosomal changes: true chromatid breaks with larger or smaller dislocations of the broken fragment of one chromatid, and gaps - achromatic regions on chromatids. The latter are not a true damage to the genome, but most often merely a change in the condensation of the protein part of chromosomes, while DNA continuity is preserved. Galloway found 0.64 % of chromatid deletions in the range of O to 6 and chromatid exchanges in 0.11 %.'' In 7000 examined metaphases of the con- /ncitlence ofspoiitaiieous cytogenetic changes in periphera/ blood /ymphocytes ofa human popu/ation .sample ,303 trol population, Karačic et al. (1995) found 0.48 % chromatid breaks, 0.27 % isochromatid chromosome breaks and 0.23 % acentric fragments. Dicentric and centric rings were not found.11 Our dala with 0.837 % chromatid breaks per subject not including gaps is comparable to the data of other authors. At the same time, these values can serve as a good indicator of the conditions of in , itro cultivation. This type of damage differs lor afferent authors, since it can be caused by the conditions of in vitro cultivation, including the qual-i y of culture medium, serum, temperature, centrif-i gation, etc. Isochromatid-chromosomal breaks, whose presence in the first in vitro division indi-< ates G I damage, was found in 0.248 % of the student group and in 0.5787 % of the technician group. The difference between the two groups is significant (p= 0.00) and may indicate the influence of age. It does not only include physiological differences caused by age, but especially lifestyle factors, which are expressed at older age. With all the simplicity and quickness of the technical procedure, as well as the possibility of machine (automatic) processing, the rnicronucleus test as the universal indicator of exposure to genotoxic substances shows a large variability in the number of micronuclei per 500 or 1000 analysed cells prepared according to the same protocol.14 Certain authors found 20 or more rnicronuclei per 1000 CB lymphocytes, while others stale 3 to 5 per thousand.5''15 This non-uniformity of data and the resulting non-availability of universal reference values dictate the need for collecting one's own data for the general population. Our results per 59000 CB cells indicate the frequency of micronuclei of 5.815 ± 2.132 (CB cells). This dala is therefore the background value lor the professionally non-exposed Slovene population. The reference value for the SCE test which is nowadays regularly used in all population rnutage-netic studies is 6.52 ± 0.702 per rnetaphase for professionally non-exposed Slovenian population, which is similar to data stated by numerous authors.51"17 No significant differences in the incidence of SCE per rnetaphase with regard to age were noticed. This test which can be considered as a mutagenetic method of choice lor the detection of exposure to chemical genotoxic agents showed very small individual deviations in our study. Por this reason it is considered to be a reliable indicator for the assessmenl of combined exposure to chemical and physical agents. The stated values of mutagenetic tests carried out on 350 test subjects provide a goocl orientation value lor mutagenetic monitoring and large ecological studies, or for the analysis of specific groups professionally exposed to genotoxic agents. Acknowledgement We gratefully acknowledge the assistance of Dr. Horvat Durda in the establishment of the Mutagenetic Unit of the Laboratory at the RS Institute for Occupational Safety, and especially for her help in the preparation of this paper. Her knowledge and rich experience are immensely appreciated, and we are especially grateful for her contribution to the pioneering work in mutage-netic monitoring in Slovenia. References 1. Carrano AV, Natarajan AT. Considerations lor population monitoring using cytogenetic techniques. ICPEMC Publication No. 14, Mutation Res 1988; 204:179-204. 2. Biological Dosimetry: Chromosomal aberration analysis for close assessment. IAEA. TRS No. 260, Vienna 1986. 3. Bender MA. Preston RJ. 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