A Study on Differences between Radiation-induced Micronuclei and Apoptosis of Lymphocytes in Breast Cancer Patients after Radiotherapy Mahnaz Taghavi-Dehaghania,*, Shahla Mohammadia,Tahereh Ziafazelia, and Manouchehr Sardari-Kermanib a Radiation Molecular Genetic Lab., National Radiation Protection Department (NRPD), Iranian Nuclear Regulatory Authority (INRA), P.O. Box 14155-4494, Tehran-14374, Iran. Fax: +982188009502. E-mail: [email protected] b Department of Radiotherapy, Cancer Institute, P.O. Box 13145-158, Tehran, Iran *Author for correspondence and reprint requests Z. Naturforsch. 60c, 938Ð942 (2005); received December 28, 2004/May 9, 2005 Cancer patients’ responses to radiotherapy vary in severity. It has been suggested that it may be due to differences in intrinsic cellular radiosensitivity. Prediction of tissue reactions to radiotherapy would permit tailoring of dosage to each patient. Towards this goal the micronucleus and apoptosis tests have been proposed as methods for measurement of chro- mosomal damage in peripheral blood lymphocytes. In this study, gamma-ray sensitivity of cultured lymphocytes of 26 breast cancer patients with early or late reactions was investi- gated. After irradiation with 4 Gy gamma radiation in G0, the frequency of micronuclei for patients with early reactions was significantly higher (P < 0.05) than for patients with late reactions. In the contrary the frequency of apoptosis for patients with early reactions was significantly lower (P < 0.05) than in the other group. It could be suggested that such a reduced amount of micronuclei in the late effects group is due to the presence of some residual DNA damages which are not completely repaired and lesions show increasing sever- ity when the patients’ cells are irradiated again. These induced damages, probably are high enough to stimulate other endpoints like apoptosis instead of micronuclei. Key words: Breast Cancer, DNA Damage, Radiosensitivity Introduction development of a simpler system of measuring chromosome damage. Schmid and Heddle pro- Cancer patients’ responses to radiotherapy vary posed independently that measuring micronuclei in severity (Tucker et al., 1992; Turesson et al., is an alternative and simpler approach to assess 1996). A range of normal tissue reactions is seen chromosome damage in vivo (Schmid, 1975; in patients, from very mild to extremely severe and Heddle, 1973). Micronuclei are discrete round occasionally lethal (Turesson and Thames, 1989). bodies of nuclear origin found in the cytoplasm A major determinant of normal tissue reaction is outside the main nucleus. They are expressed in the intrinsic radiosensitivity of the cells (Tures- dividing cells that either contain chromosome son, 1990). breaks lacing centromeres (acentric fragments) Much of radiobiology is concerned with increas- and/or whole chromosomes that are unable to ing our understanding of why and how normal travel to the spindle poles during mitosis. At telo- tissues and tumors respond to radiation with the phase, a nuclear envelope forms around the lag- ultimate aim of improving cancer treatment by ra- ging chromosomes and fragments, which then un- coil and gradually assume the morphology of an diotherapy. Hence the need for reliable predictive interface nucleus with the exception that they are assays for normal tissues and tumors response to smaller than the main nuclei in the cell, hence they radiation remains a prime objective of clinical on- are “micronuclei”. In 1985 Fenech and Morley cology. found that the addition of cytochalasin B (cyt B) The micronuclei assay and apoptotic cell death to the culture medium prevents cells from com- are biological indicators for assessment of radio- pleting the division cycle by inhibiting cytokinesis sensitivity (Muller et al., 1996; Hendry and West, but not karyokinesis. By using this technique, cells 1997). with only one mitotic division since the addition The complexity and laboriousness of enumerat- of cyt B included two nuclei (Fenech and Morley, ing aberrations in metaphases has stimulated the 1985a, b). 0939Ð5075/2005/1100Ð0938 $ 06.00 ” 2005 Verlag der Zeitschrift für Naturforschung, Tübingen · http://www.znaturforsch.com · D M. Taghavi-Dehaghani et al. · Micronuclei and Apoptosis in Breast Cancer 939 Physiological cell death was described initially 2000). Following the investigations by Jones et al. based on morphological grounds only, and was (1995), we compared the sensitivity of lympho- given the name apoptosis. Morphological changes cytes of breast cancer patients with early/late reac- at the microscopic level associated with apoptosis tions after radiotherapy by studying the radiation are condensation of chromatin into crescent- induced micronuclei and apoptosis frequency. This shaped caps at the nuclear periphery, disintegra- study could be used as a test to predict clinical tion of the nuclei, and reduction in nuclear size, radiation reactions. shrinkage of total cell volume, budding (blebbing) and constriction of both the nucleus and the cyto- Materials and Methods plasm into multiple, small, membrane-bound apoptotic bodies (Kerr et al., 1972). Blood samples Peripheral blood lymphocytes are ideal cells for Heparinzed peripheral blood samples were ob- measuring normal tissue radiosensitivity, since tained from 26 breast cancer patients with early or they are easy to obtain and to use for in vitro tests. late reactions aged between 36 and 72 years. Fif- Lymphocytes are particularly susceptible to DNA teen patients had shown early tissue damage (ery- damage-induced agents because of their high po- thema, dry desquamation, moist desquamation) tential for mutation. They undergo DNA damage and eleven patients had shown late tissue damage in a time- and dose- dependent manner. Because (fibrosis, skin telangiectasia, pigmentation). Pa- of the kinetics and the relative sensitivity of lym- tient characteristics and treatment parameters are phocytes to low doses of radiation, they may be a presented in Table I. Lymphocytes were separated useful biological dosimeter for radiation exposure. from whole blood on Ficoll-Hypaque gradients On the other hand they are a synchronous cell (AXIS-SFIELD poC AS Oslo, Norway), washed population, so that the synchrony would obviate twice in phosphate buffered saline (PBS) and re- the difference caused by mixing of cells treated at suspended in RPMI 1640 (Gibbco BRL) medium different stages of the cell cycle (Evans and O’Ri- modified with l-glutamine containing 10% heat- ordan, 1975; Catena et al., 1996; Boreham et al., inactivated fetal calf serum (Gibbco BRL), 100 U a b c Table I. Patient characteristics and Number Age Path GD Days Days Side treatment parameters. of radd of cheme effects 150Dg 5000 25 8 Late 236LOh 5000 25 6 Early 344D5000 25 3 Late 442LO5000 25 6 Early 548D5000 25 6 Early 665D5000 25 Ð Early 739D5000 25 6 Late 846D5000 25 6 Early 950D5000 25 6 Early 10 38 D 5000 25 6 Early 11 62 D 5000 25 6 Early 12 44 D 5000 25 6 Early 13 68 D 5000 25 9 Early 14 43 D 5000 25 6 Early 15 53 D 5000 25 6 Early 16 72 D 5000 25 6 Late 17 53 D 5000 25 Ð Late 18 41 D 3000 25 6 Late 19 44 D 5000 25 6 Late i Ð 20 51 I 5000 25 Early a 21 41 D 5000 21 6 Early Age in years at time of blood sam- pling; b histopathological type of tumor; 22 46 D 5000 30 6 Late c d 23 62 D 5000 25 6 Early given dose in Rad; number of days 24 46 D 5000 25 6 Late that patients were treated in radiother- apy; e number of days that patients 25 47 D 5000 25 6 Late g 26 71 D 5000 30 2 Late were treated in chemotherapy; D, duc- tal; h LO, lobular; i I, in situ. 940 M. Taghavi-Dehaghani et al. · Micronuclei and Apoptosis in Breast Cancer mlÐ1 penicillin and 100 µgmlÐ1 streptomycin tive was used to resuspend the cells pellet and (Boehringer Mannheim GmbH, Germany). approx. 50 µl were spread onto a microscope slide. The slides were air-dried and stained in 10% Irradiation Giemsa. The apoptotic cells were scored in 1000 cells with the light microscope using a 1000x mag- Each sample was divided into two parts, one part nification. was used as a control and the other part was irradi- ated with 4 Gy gamma radiation using a 60Co source (Gamma beam 150 Atomic Energy, Canada). Statistical analysis Student’s test was performed to assess the dif- Micronuclei assay ferences between experimental groups. Samples were kept for 1 h at 37 ∞C after being irradiated. The cells were then cultured at a con- Results 6 Ð1 centration of 10 cells ml at 37 ∞Cinahumidi- Micronucleus frequency µ fied atmosphere containing 5% CO2 adding 15 g mlÐ1 phytohaemagglutinin-M (Boehringer Mann- The measurement of frequency of micronucle- heim GmbH) to stimulate proliferation. After 44 h ated binucleate lymphocytes and the total of mi- incubation, 4.0 µgmlÐ1 Cyt-B (Sigma) were cronuclei in 1000 binucleate lymphocytes showed added, and cells were harvested at 72 h. The cells the following results: were treated with a lypotonic solution containing Irradiation induced more multimicronuclei in 0.075 m KCl for 4Ð6 min, to obtain good preserva- binucleated lymphocytes (tri-quadric- or more mi- tion of the cytoplasm. Following centrifugation at cronuclei were formed in irradiated samples). 16.1 ¥ g for 5 min, the cells were fixed in 3:1 (v/v) Irradiation has induced more multimicronuclei methanol/acetic acid. Afterwards, the cells were in the early group than the late group, suggesting dropped onto cooled slides with a Pasteur pipette that more sever damage had occurred. The mean and air-dried. The slides were stained in 10% of binucleate cells with micronuclei and the mean Giemsa (Merck) and the MNs were scored in 1000 of the total micronuclei in binucleate cells for pa- binucleate cells. They were observed by a light mi- tients with early reactions were significantly higher croscope using a 400x magnification based on the (P < 0.05) than for patients with late reactions criteria summarized by Fenech (2000).