Radiol Oncol 2006; 40(1): 43-9. Phytohaemagglutinin as a modulator of DNA repair measured by chromosome aberration analysis in micronucleus assay in ionizing radiation biodosimetry Martina Đurinec, Davor Želježić and Vera Garaj-Vrhovac Institute for Medical Research and Occupational Health, Mutagenesis Unit, Zagreb, Croatia Background. There are some correlations between cell’s ability to remove DNA damage and proliferative activity. The aim of this study was to examine the influence of phytohaemagglutinin (PHA) on DNA repair capacity in isolated human lymphocytes exposed to ionizing radiation. Methods. Lymphocytes were isolated from the whole blood using a Ficoll centrifugation. As the source of ?-rays 60Co source Alcon, CGR-MeV was used. To achieve the absorbed dose of 2 Gy a total exposure to radiation lasted for 1.24 minutes at room temperature. Possible differences in DNA repair efficiency were monitored by chromosomal aberration analysis and micronucleus assay, 48 and 72 h after the PHA stimulation, respectively. Results. The number of dicentric chromosomes and acentric fragments were significantly increased in lym-phocytes stimulated by phytohaemagglutinin immediately after the irradiation compared to the cultures where the activator was added after 1, 2 and 4 h. The micronucleus assay did not show any significant dif-ferences in the number and distribution of micronuclei regardless of the time when the mitogen activator was added. Conclusions. The observed non-significant decreases in the total number of chromosomal aberration and micronuclei suggest that phytohaemagglutinin does not significantly contribute to the DNA repair. Key words: ionizing radiation; DNA damage; DNA repair; phytohaemagglutinins; chromosome aberra-tions; micronucleus test Received 10 January 2006 Accepted 9 February 2006 Correspondence to: Martina Đurinec, BSc, Institute for Medical Research and Occupational Health, Mutagenesis Unit, Ksaverska cesta 2, Zagreb, Croatia. Phone: +385 1 467 31 88; E-mail: djurinec@imi.hr Introduction During the last few decades ionizing radiation became unavoidably present in human lives. It is widely used in the variety of med-ical diagnostic procedures as electric energy source of nuclear power plants. It affects a human organism on daily basis in the form of cosmic radiation. Since air flights are becoming more common way of travelling, and it is 44 Đurinec M et al. / Phytohaemagglutininas a mediator of DNA repair known that cosmic radiation is significantly higher in upper layers of the atmospheres, the total dose population is expected to in-creases. It is known that ionizing radiation (IR) deposits its energy in cellular structures through discrete ionization events that are es-sentially randomly distributed in space. Unlike chemical agents, whose damaging potential is strongly dependent on diffusion processes and thus may be affected by sub-cellular structures, IR is highly penetrating: the physics and subsequent chemistry associated with the photon absorption and the ion-izing events that occur along fast electron track are complete within a few microsec-onds.1 Ionizing radiation causes a wide spectrum of chemically different types of lesions in DNA of which the so-called locally multi-ply damaged sites (LMDS) are assumed to be biologically most important.2,3 LMDS may consist of single-strand breaks (SSB) on op-posite strands that, if located close to each other, may give rise to double strand breaks (DSB).4 Thus, DSB induced by IR may arise as a direct consequence of one or more ionizing events or indirectly as a base or sugar damage on opposite strands. Ionizing radiation in physicochemical interaction with cellular DNA also produces a variety of primary lesions, alkali-labile sites, DNA-DNA and DNA- protein crosslinks, and damage to purine and pyrimidine bases.5-8 DNA double strand breaks (DSB) are the most serious form of DNA damage. There are two path-ways for the repair of DSB.9 One is homolo-gous recombination (HR) which occurs dur-ing late S and G2 phase of the cell cycle and the other pathway is non homologous DNA end joining (NHEJ). It is predominant during G0, G1 and S phase of the cell cycle.10 If not repaired, DNA lesions could cause cell death. If misrepaired, DSBs contribute to chromosomal aberrations and genomic instability. Ionizing radiation produces chromosome aberrations (involving two chromatids) at the S phase and the chromatid aberrations at G2. Radiol Oncol 2006; 40(1): 43-9. To detect genetic alterations at the chromo-some level using chromosome aberration and micronucleus assay the cells should be in-duced to enter the G1 phase and undergo di-vision.11-14 Phytohaemagglutinin (PHA) selectively stimulates T lymphocytes to enter mitosis. The widespread popularity of peripheral blood culture as means of chromosome analysis has been largely dependent on that mitogen.15-23 There is some correlation be-tween cell’s ability to remove the DNA dam-age and its proliferative activity. It is well es-tablished that unstable aberrations like dicen-tric chromosomes, chromosome breaks and acentric fragments can be eliminated during the cell division. It was suggested that the regulation of DNA repair is dependent on cell cycle. It involves the expression of DNA re-pair enzymes within the defined program of gene control during the cell cycle. Some au-thors have shown that immediately after the irradiation mitogen-stimulated cells have a higher frequency of chromosome aberrations than the cells resting in G0 phase before the addition of mitogens.15 Our study aimed to examine the influence of PHA on DNA repair capacity of isolated human lymphocytes. Cell cultures were started and PHA was added 0, 1, 2 and 4 h after the irradiation. Possible differences in the DNA repair efficiency was monitored by chromosomal aberration analysis and mi-cronucleus assay, 48 and 72 h after the PHA stimulation, respectively. Methods Isolation of lymphocytes The whole blood sample was taken from the cubital vein of a healthy adult male volunteer using heparinized vacutainer (Becton Dickinson, USA). There is no record that prior to the study the volunteer was exposed to any physical or chemical agent that might in- Đurinec M et al. / Phytohaemagglutininas a mediator of DNA repair 45 terfere with the results. Lymphocytes were isolated from the whole blood sample by a Ficoll centrifugation method.24 One milliliter of the whole blood was resuspended in 8 ml of Ham’s F-10 essential medium supplement-ed with L-glutamine, bovine serum (20%), penicillin (100 I.U./ml) and streptomycin (100 µg/ml). Irradiation of isolated lymphocyte As the source of ?-rays 60Co source Alcyon, CGR-MeV was used. Vacutainer containing isolated lymphocytes was mounted in an acrylic phantom (dimensions: 20x20x15 cm3), in depth of 5.5 cm, transversally to the axis of the irradiation. The radiation field was 15 x 15 cm2, and the distance between the surface of phantom and the source of radiation was 80 cm. At total exposure to radiation lasted for 1.24 min at room temperature, thus the absorbed dose was 2 Gy. Cultivation of lymphocytes Phytohaemagglutinin (Murex Biotech Ltd.) (0,2ml) was added to lymphocyte cultures ei-ther immediately after the irradiation or after a certain recovery period (1, 2 or 4 h). Meanwhile cultures were held at 37°C.13 Since the analysis was done in duplicate, for each stimulation time, 4 different cultures were started: for the chromosomal aberration analysis and for the micronucleus assay. The analysis of structural chromosome aberrations The structural chromosome aberration analy-sis test was performed according to current IAEA guidelines.25 Simultaneously with cul-tures for the micronuclei assay, cultures for the chromosome aberration test were set up in the same manner. Duplicate cultures per sample were set up and incubated at 37°C for 48 h. After the PHA stimulation to arrest di-viding lymphocytes in metaphase, colchicine (Sigma) (0.004%) was added 2 h prior to the harvest. Cultures were centrifuged at 1000 rpm for 10 min, the supernatant was care-fully removed, and the cells were resuspend-ed in a hypotonic solution (0.075M KCl) at 37°C for 20 min. After the second centrifu-gation, the cells were fixed with a freshly prepared fixative of ice-cold methanol/gla-cial acetic acid (v/v 3:1). Fixation and cen-trifugation were repeated several times until the supernatants were clear. The cell suspension was dropped onto microscope slides and left to air-dry. Slides were stained with 5% Giemsa solution (Merck). For each stimulation analysis was done in duplicate, a total number of 200 methaphases was scored. Structural chromosome aberrations were classified based on the number of sis-ter chromatids and breakage events in-volved. Only metaphases containing 46 cen-tromeres were analyzed. A total number of each type of aberrations, as well as the per-centage of aberrant cells per subject were evaluated. Micronucleus assay The micronucleus assay was performed as described by Fenech and Morley with some modifications.26 After the irradiation lympho-cyte cultures were set up by adding 1 ml of isolated lymphocytes to 8 ml of F-10 medium (Sigma) supplemented with foetal calf serum (Sigma) and antibiotics penicillin (Pliva) and streptomycin (Krka). Following the stimulation with PHA, lymphocytes were incubated in vitro for 72 h at 37°C. Cytochalasin-B (Sigma) at the final concentration of 6 mg/ml was added to each culture at 44 h, and the cells were harvested after a further incubation of 28 h. After the treatment with physiological saline, cells were fixed with cold fixative, a mixture of methanol: acetic acid (v/v 3:1). The fixation step was repeated twice and cells were resuspended in a small volume of fixa-tive solution and dropped onto clean slides. Radiol Oncol 2006; 40(1): 43-9. 46 Đurinec M et al. / Phytohaemagglutininas a mediator of DNA repair Finally, they were stained with 5% aqueous solution of Giemsa dye (Merck) for 10 minutes. For each stimulation the analysis was done in duplicate, thus a total of 500 binu-clear lymphocytes were scored. The data are expressed as the number of micronuclei per 500 binucleated cells as well as the frequency of binucleated cells containing one or more micronuclei. Statistical analysis The statistical significance of the results ob-tained was evaluated using the ?2 - test. The level of statistical significance was set at 5%. Chi-Square test was used to compare the fre-quencies of chromosomal aberration and mi-cronuclei. Results Chromosomal aberration analysis The number of dicentric chromosomes and acentric fragments was found to be signifi-cantly increased in all irradiated lymphocytes regardless of the start point of the PHA stimulation compared to the control (p<5%). In irradiation exposed sample there were 97 acen-tric fragments and 12 dicentrics observed in PHA stimulated lymphocytes whereas in the control no dicentrics and 4 acentic fragments were found. The number of aberrations be-tween cultures stimulated 1, 2 and 4 hours after the irradiation did not differ significantly (p< 5%). In cultures stimulated with PHA 1, 2 and 4 hours after the irradiation the number of acentric chromosomes and dicentric chromo-somes significantly decreased compared to the cultures where mitogen was added imme-diately after the irradiation. One hour after the irradiation the number of acentrics was 57, two hours after the irradiation the number of acentric chromosomes was 69 and four hours after the irradiation the number of acentrics was 48. The number of dicentric chromosomes decreased compared to the number of acentric chromosomes. Immedia-tely after the irradiation the number of dicen-tric chromosomes was 12, one hour after the stimulation the number of dicentrics was 1. Still, the difference in the number of di-centrics was not found to be significant (Table 1). Micronucleus assay Using micronucleus assay no significant dif-ferences in number of micronuclei were ob- Table 1. Total number and distribution of chromosome aberrations in isolated human lymphocytes stimulated to proliferate after the indicated post-irradiation periods. Two hundred cells were analyzed per each PHA stimulation point. Time after Total number irradiation of abberations Chromatid breaks Chromosome breaks Acentric fragments Dicentric chromosomes %of cells with abberations Isolated lymphocytes irradiated with 2 Gy 0 h 112a 3 / 97a 12a 38.0 1 h 58a,b / / 57a,b 1 21.5 2 h 76a,b 1 1 69a,b 5 29.0 4 h 53a,b / / 48b 5 23.0 Non-irradiated samples Lymphocytes 4 / / 4 / 2.0 Whole blood 2 / / 2 / 1.0 astatistically significant compared to the control P < 0.05 bstatistically significant compared to the 0h PHA stimulation point P < 0.05 Radiol Oncol 2006; 40(1): 43-9. Đurinec M et al. / Phytohaemagglutininas a mediator of DNA repair 47 Table 2. Frequencies of micronuclei in binucleated human lymphocytes stimulated to proliferate after the indicated post-irradiation periods. Five hundred cells were analyzed per each PHA stimulation point. Time after Cells Cells S MN / Distribution of micronuclei MN / irradiation without MN with MN 500 cells 1 MN 2 MN 3 MN 4 MN Cell Isolated lymphocytes irradiated with 2 Gy 0 h 396 104 116 94 8 2 / 0.23 1 h 411 89 104 74 15 / / 0.21 2 h 399 101 117 89 9 2 1 0.23 4 h 411 89 95 83 6 / / 0.19 Non-irradiated samples Lymphocytes 499 1 1 1 / / / 0.002 Whole blood 498 2 3 1 1 / / 0.006 MN = micronuclei served, regardless of the time mitogen activa-tor was added. In irradiated cultures the num-ber of micronuclei per cell ranged from 0.19-1.23 compared to the control where it was 0.002. Discussion This study presented the possible influence of phytohaemagglutinin on DNA-repair scor-ing the number of chromosome aberrations and micronuclei. To initiate DNA damage lymphocytes were irradiated with 2 Gy 22,23 using a ?-ray 60Co source. The number of acentric fragments was significantly in-creased in lymphocytes stimulated by phyto-haemagglutinin immediately after the irradiation compared to the cultures where the acti-vator after 1, 2 and 4 h was added (Table 1). That finding could indicate that DSB repair mechanisms are efficient in G0 phase of the cycle and/or that the stimulation of lympho-cytes to undergo division without having time to eliminate the majority of the DNA lesions in G0 phase increases a misrepair rate result-ing in the increased number of chromosome type aberrations. These results support the finding that the formation of unstable aberra-tions is cell cycle dependent and that most of double strand breaks can be fixed in first 24 h after the irradiation.27 The same result but with different ap-proach was observed by Mayer et al. They showed that a higher level of the DNA repair events in stimulated cells does not necessarily reflect a higher DNA repair capacity. Additionally, they showed that all repair proteins needed for the repair of ?-irradiation in-duced DNA-damage are already present in G0 cells at sufficient amounts and do not need to be induced once lymphocytes are stimulated to start cycling.28 Only specific DNA repair genes were found to be up-regulated after the PHA stimulation of which most have an addi-tional function in the DNA replication. The mitogen stimulation of lymphocytes may re-sult in an increased removal of only specific types of DNA lesions as it was reported by other authors.29,30 This observation might be explained by the cell cycle dependent regulation of specific DNA repair enzymes, that are more active in proliferating than in resting cells or by differences in the availability of de-oxyribonucleotides which are necessary for the DNA excision repair which is not involved in DSB repair.30,31 Mayer et al.28 identified on-ly 12 genes that responded with a more than 2-fold increase of transcripts to the mitogenic stimulus, with a maximum induction for each of the genes 72 h after the PHA treatment. A decrease in the number of chromosome type aberrations with the delay of PHA stimuli could indicate the gradual activation of addi-Radiol Oncol 2006; 40(1): 43-9. 48 Đurinec M et al. / Phytohaemagglutininas a mediator of DNA repair tional repair capacities, but still the decrease was not to found to be significant. That obser-vation is in the correlation with findings that more than 70% of all evaluated genes had con-stant expression levels within a twofold range compared to unstimulated.28 As shown in the Table 2. no significant dif-ferences were observed in the number of mi-cronuclei, regardless of the time point when the mitogen activator was added. Neverth-eless, the number of micronuclei for specific PHA stimulation point was significantly higher than the number of chromosomal type of aberrations. It indicates that all micronuclei formed do not originate from acentrics only but also from entire chromosomes.32 Micro-tubules remain unsorted within the mitotic plane forming micronuclei.11 Obtained results could indicate that the repair of those lesions is not dependent on the time passed between the irradiation and the mitogen stimulation. Our results show the baseline level of fre-quency of micronuclei after 3 cell cycles which is the same as Ramirez et al. observed.32 The observed non-significant decreases in the total number of chromosomal aberration and micronuclei suggest that phytohaemagglu-tinin does not significantly contribute to the DNA repair. We could say that in order to maximize the sensitivity of the chromosomal aberration analysis phytohaemagglutinin has to be added immediately after the irradiation. References 1. Pfeiffer P, Goedecke W, Obe G. Mechanisms of DNA double-strand break repair and their potential to induce chromosomal aberrations. Muta-genesis 2002; 15: 289-302. 2. Ward JF. DNA damage produced by ionizing radiation in mammalian cells: identities, mechanisms of formation and reparability. Prog Nucleic Acid Res Mol Biol 1998; 35: 95-125. 3. Ward JF. The yield of DNA double-strand breaks produced intracellularly by ionizing radiation: A review. Int J Radiat Biol 1990; 57: 1141-50. 4. Friedberg EC, Walker GC, Siede W. DNA Repair and Mutagenesis. In: Gerlach VL, Aravind L, editors. Biological Responses to DNA Damage. Cold Harbor: Laboratory Press Inc., 2001. p. 8-18. 5. Garaj-Vrhovac V, Kopjar N. The alkaline Comet assay as biomarker in assessment of DNA damage in medical personnel occupationally exposed to ionizing radiation. Mutagenesis 2003; 18: 265-71. 6. Garaj-Vrhovac V, Kopjar N, Ražem D, Vekić B, Miljanić S, Ranogajec-Komor M. Application of the alkaline comet assay in biodosimetry: assess-ment of in vivo DNA damage in human peripher-al leukocytes after a ? radiation incident. Radiat Prot Dos 2002; 98: 407-16. 7. Natarajan AT. Mechanisms of induction of muta-tions and chromosome alterations. Environ Health Perspect Suppl 1993; 101: 225-9. 8. Wilson VL, Taffe BG, Shields PG, Povery AC, Harris CC. Detection and quantification of 8 hy-droxydeoxyguanosine adducts in peripheral blood of people exposed to ionizing radiation. Environ Health Perspect 1993; 99: 261-3. 9. Liang F, Han M, Romanienko PJ, Jasn M. Homology-directed repair is a major doublestrand break repair pathway in mammalian cells. Proc Natl Acad Sci 1998; 95: 5172-7. 10. Sonoda E, Sasaki MS, Buerstedde JM, Bezzubova O, Shinohara A, Ogawa H, et al. Rad 51-defined vertebrate cells accumulate chromosomal breaks prior to cell death. EMBO J 1998; 11: 598-608. 11. Maluf SW. Monitoring DNA damage following radiation exposure using cytokinesis-block micronu-cleus method and alkaline single-cell gel elec-trophoresis. Clin Chim Acta 2004; 347: 15-24. 12. Buckton KE, Hamilton GE, Paton L, Langlands AG. Chromosome aberrations in irradiated anky-losing spondylitis patients. In: Evans H, Lloyd DC, eds. Mutagen-induced chromosome damage in man. London: Edinburgh University Press Inc., 1978. p.142-5. 13. Natarajan AT, Obe G. Screening human popula-tions of mutations induced by environmental pol-lutants: use of human lymphocyte system. Ecotoxicol Environ Saf 1980; 4: 468-81. 14. Carrano AV, Natarajan AT. Considerations for population monitoring using cytogenetics tech-niques. ICPEMC publication 14. Mutat Res 1988; 204: 379-406. 15. Santos Mello R, Kway D, Norman A. Chromosome aberrations and T-cells survival in human lympho-cytes. Radiat Res 1974; 60: 482-8. Radiol Oncol 2006; 40(1): 43-9. Đurinec M et al. / Phytohaemagglutininas a mediator of DNA repair 49 16. Arakaki D, Sparkes RS. Microtechnique for cul-turing lekocytes from whole blood. Cytogenetics 1963; 2: 57-60. 17. Li JG, Osgood EE. A method for the rapid separation of leukocytes and nucleated erythrocytes from blood or marrow with a phytohaemagglu-tinin from red beans (Phaseolus vulgaris). Blood 1949; 4: 670-5. 18. Maluish AE, Strong DM. Lymphocyte proliferation. In: Rose NR, de Macario EC, Folds JD, Lane HC, Nakamura RM, editors. Manual of clinical im-munology. Washington: DC Press Inc., 1986. p. 274-81. 19. Nowell PC. Phytohaemagglutinin: an initiator of mitosis in cultures of normal human leukocytes. Cancer Res 1960; 20: 462-6. 20. Waithe Wi, Hirschhorn K. Lymphocyte response to activators. In: Weir DM, editor. Handbook of ex-perimental immunology. Oxford: Blackwell Scien-tific Publications, 1979. pp. 26.1.-26.10. 21. Watt JL, Stephen GC. Lymphocyte culture for chromosome analysis. In: Rooney DE, Czepul-kowski BH, eds. Human cytogenetics- A practical ap-proach. Oxford, UK: IRL Press Inc., 1986. p.39-55. 22. Savoldi-Barbosa M, Sakamoto ET, Takahashi CS. Influence of novobiocin on g-Irradaited G0-lym-phocytes as analysed by cytogenetic endpoints. Gen Mol Biol 1999; 22: 217-23. 23. Boerrigter Meti, Vijg J. Single strand break disap-pearance in quiescent and phytohaemagglutinin-stimulated human peripheral blood lymphocytes exposed to a single low dose of ?-irradiation. Int J Radiat Biol 1992; 61: 95-101. 24. Obe G, Beek B, Dudin G. The human leukocyte test system: V. DNA synthesis and mitoses in PHA stimulated 3-day cultures. Humangenetik 1975; 28: 295-302. 25. International Atomic Energy Agency -IAEA. Cytogenetic analysis for radiation dose assess-ment. Technical Reports Series. Vienna; 2001; No 405. 26. Fenech M, Chang WP, Kirsch-Volders M, Holland N, Bonassi S, Zeiger E. HUMN project: detailed description of the scoring criteria for the cytokine-sis-block micronucleus assay using isolated human lymphocyte cultures. Mutat Res 2003; 534: 65-75. 27. Rothkamm K, Lobrich M. Misrepair of radiation -induced DNA double-strand breaks and its rele-vance for tumorigenesis and cancer treatment. Int J Oncol 2002; 121: 433-40. 28. Mayer C, Popanda O, Zelezny O, von Brevern MC, Bach A, Bartsch H, et al. DNA repair capacity after gama irradiation and expression profiles of DNA repair genes in resting and proliferating human peripheral blood lymphocytes. DNA Repair (Amst) 2002; 1(3): 237-50. 29. Sirover M. Cell cycle regulation of DNA repair en-zymes and pathways. In: Transformation of human diploid fibroblasts. Boca Raton, Florida: CRC Press Inc, 1990. p. 29-54. 30. Boerrigter Meti, Mullaart E, Vilg J. Induction and disappearance of DNA strands breaks in human peripheral blood lymphocytes and fibroblasts treated with methyl methanesulfonate. Exp Cell Res 1991; 192: 61-6. 31. Green MHL, Waugh APW, Lowe JE, Harcourt SA, Cole J, Arlett CF. Effect of deoxyribonucleotides of human peripheral blood lymphocytes on the hy-persensitivity of human peripheral blood lympho-cytes to UV-B and UV-C irradiation. Mutat Res 1994; 315: 25-32. 32. Ramirez MJ, Surralles S, Puerto S, Creus A, Marcos R. Low persisrtance of radiation-induced centromere positive and negative micronuclei in cultured human cells. Mutat Res 1999; 440: 163-9. Radiol Oncol 2006; 40(1): 43-9. Slovenian abstracts 61 Radiol Oncol 2006; 40(1): 39-42. Erlotinib pri zdravljenju nedrobnoceličnega raka pljuč Smrdel U, Kovač V Izhodišča. Erlotinib je novo biološko protitumorsko zdravilo, ki ga uporabljamo pri bolnikih z nedrobnoceličnim rakom pljuč. Predstavlja molekularno tarčno terapijo, ki so jo zelo intenzivno proučevali v kliničnih študijah. Prikaz primera. Predstavljamo bolnika, ki smo ga obravnavali zaradi razširjene oblike nedrob-noceličnega raka pljuč. S kemoterapijo smo dosegli stagnacijo bolezni, ki je po desetih mesecih ponovno napredovala. Splošno stanje se je izrazito poslabšalo. Že po enem mesecu ponovnega sistemskega zdravljenja, tokrat z erlotinibom, so simptomi izzveneli in na rentgenski sliki prsnih organom vidimo znatno zmanjšanje bolezenskih sprememb (delno remisijo). Zaključki. Pri izbranih bolnikih z nedrobnoceličnim rakom pljuč lahko erlotinib podaljša preživetje in znatno zmanjša simptome bolezni. Radiol Oncol 2006; 40(1): 43-9. Fitohemaglutinin kot modulator DNK popravljalnih mehanizmov merjenih s številom kromosomskih aberacij in z mikronukleus testom po obsevanju z ionizirajočim sevanjem Đurinec M, Želježić D in Garaj-Vrhovac V Izhodišča. Obstaja korelacija med sposobnostjo popravila poškodb DNK in sposobnostjo delitve celic. Zato je bil namen študije ugotoviti vpliv fitohemaglutinina (PHA) na sposobnost popravila DNK poškodb pri izoliranih humanih limfocitih, obsevanih z ionizirajočim sevanjem. Metode. Limfociti so bili izolirani na Fikol gradientu. Za obsevanje celic smo uporabili 60Co izvor proizvajalca Alcon. Celice so bile obsevane 1,24 minut pri sobni temperaturi, tako smo dosegli absorbirano dozo 2 Gy. Razlike v sposobnosti popravila DNA poškodb smo merili s številom kromosomskih aberacij in z mikronukleus testom 48 in 72 ur po PHA stimulaciji. Rezultati. Število dicentričnih in acentričnih kromosomov je bilo signifikantno povečano pri celicah, ki so bile stimulirane s PHA takoj po obsevanju, ne p apri celicah, kjer smo dodali PHA 1, 2 ali 4 ur kasneje. Mikronukleus test ni pokazal signifikantnih razlik v distribuciji ne glede na čas dodanega fitohemeglutinina. Zaključek. Rezultati nakazujejo, da fitohemaglutinin ne vpliva signifikantno na popravljalne mehanizme DNK. Radiol Oncol 2006; 40(1): 57-62.