VII Radiation Physics & Protection Conference, 27-30 November 2004,Ismailia-Egypt EG0500347 The Reactivity of Plant, Murine and Human Genome to Electron Beam Irradiation L. Gavrilã*, D. Timus**, I. Radu*, D. Usurelu*, E. Manaila** *Bucharest University, Genetic Institute, Aleea Portocalilor 1-3, Tel.: +40-21-2127368; fax: +40-21-2248846, 060101-BUCHAREST, ROMANIA, email:[email protected] **National Institute for Laser, Plasma and Radiation Physics, Electron Accelerators Laboratory, Str. Atomistilor 409, 077125-BUCHAREST-Magurele, ROMANIA, email: [email protected] ABSTRACT A broad spectrum of chromosomal rearrangements is described in plants (Allium cepa), mouse (Mus musculus domesticus) and in humans (Homo sapiens sapiens), following “in vivo” and “in vitro” beta irradiation. Irradiations were performed at EAL**, using a 2.998 GHz traveling-wave electron accelerator. The primary effect of electron beam irradiation is chromosomal breakage followed up by a variety of chromosomal rearrangements i.e. chromosomal aberrations represented mainly by chromatid gaps, deletions, ring chromosomes, dicentrics, translocations, complex chromosomal interchanges, acentric fragments and double minutes (DM). The clastogenic effects were associated in some instances with cell sterilization (i.e. cell death). Key Words: Electron Beam Irradiation / Chromosomal Aberration / Clastogenic Effects INTRODUCTION The incidence and the severity of damages induced in genetic material by irradiation rise with increasing dose and are not recognized below a threshold dose (1-3). The study of the action of electron beam irradiation included different biological systems like plants, laboratory animals as well as in vivo culture of human cells. Such data bring precious information concerning the interaction of the immune processes and the repairing mechanisms of DNA lesions induced by the radiations. A primary event induced by irradiation is chromosome breakage, followed by chromosome rearrangements and genomic instability. The chromosomal aberrations are very useful biomarkers as intermediate end points in evaluating harmful biological effects of ionizing radiation. Informations about the risk of cancer in an irradiated population can also be obtained after screening the frequency of chromosomal aberrations and scoring both unstable (dicentrics, ring chromosomes and acentrics) and stable (inversions, duplications, deletions, Robertsonian translocations etc.) chromosomal rearrangements. A plethora of data on cytogenetic effects of ionizing irradiation exists for plant, mice and human, but they are presented separately (1-6). Our study tried to perform a comparative investigation in these species. Corroborating these data will finally allow the achievement of a coherent description concerning the reactivity of the human genome to radiation, with major implications for the modern therapeutic strategies, and prevention of noxious effects of irradiation to human being health. 713 VII Radiation Physics & Protection Conference, 27-30 November 2004,Ismailia-Egypt MATERIALS AND METHODS Irradiations were performed at Electron Accelerators Laboratory, National Institute for Laser, Plasma and Radiation Physics, Magurele-Bucharest, Romania. The samples were irradiated using an ALIN 10 linear electron accelerator (the later being an indigenous product of Romania). ALIN 10 is a travelling wave type linac operating at 2.998 GHz, 6.5 MeV mean energy, with a 0.1 mm Al foil exit window. Improved Fricke (ferrous sulphate, cupric sulphate and sulphuric acid in triple distilled water) dosimetry system has been used to perform preliminary dose measurements. The plant biological material is represented by Allium cepa bulbs. Irradiations have been made on sets of six onions, as follows: 0.55; 5.10; 10.35; 20.70 Gy. The root meristems of the irradiated bulbs were processed according to Feulgen method, in order to evidence the chromosomal aberrations and different clastogenic effects of irradiation. Six males of Balb stock mice (Mus musculus domesticus), provided by Cantacuzino Institute biobase in Bucharest, two months of age, were irradiated, as a whole body procedure. The irradiation doses were: 2.05; 3.10; 3.71; 4.12; 4.72; 5.15 Gy. Animals from the same inbreed line were investigated as control. As biological material femoral bone marrow was used. The conventional Hungerford method on short-term cultures for 52 hrs was adapted for human chromosome investigation. The peripheral blood was collected from aged 25, healthy, non-smoker donor, which is non-alcohol consumer and without any history of irradiation or severe long viral infection. The doses used to irradiate human blood cultures were: 1.95; 2.93; 3.42; 4.07; 4.56; 5.05 Gy. Six tubes for each dose of irradiation were used. The slides were prepared by air-drying and stained with a 10% Giemsa solution (pH=6.8). At least 100 well – spread metaphases were examined, both for each experimental variant and for control. Visualization of slides has been performed using an Amplival optic microscope, with 100x immersion objective lens. The photographs have been performed using a Praktika MTL 3 photo camera. RESULTS, DISCUSION AND CONCLUSIONS The main effect of irradiation upon genetic material has been chromosomal fragmentation. An intense chromosomal stickness, which leads to atypical chromosomal associations (Figs. 4, 5, 7, 8) was noticed, in many cells of A. cepa. This led to atypical chromosomal clumps, sometimes forming long rows (Figs. 7, 8). The dicentric chromosomes (Fig. 5) resulted from telomere-to- telomere and telomere-to-centromere fusions. Numerous ring chromosomes have also been encountered (Fig. 6) (Plate I, Figs. 1-8). Types of structures such as double minutes, (seldom present in plants) (Fig. 7) have been generated as a consequence of amplification under abnormal conditions. The acentric fragments generated by terminal deletions, which affect a single or, more rarely, both chromatides of a metaphase chromosome, have also been noticed, usually associated with chromosomal translocations (Fig 8). The appearance of chromatic bridges (Fig. 1) and micronuclei (Fig. 2) is a common sign of abnormal cell division. (Table 1) 714 VII Radiation Physics & Protection Conference, 27-30 November 2004,Ismailia-Egypt Table 1 ONION CHROMOSOMAL MODIFICATION (Gy) (Gy) Irradiation doses Abnormal metaphases Double minutes Ring chromosomes Micronuclei Dicentrics Chromatic bridges Acentric fragments Total Control (0) 3 0 0 0 0 0 1 1 0.55 (1) 5 0 0 1 1 1 2 5 5.10 (2) 10 2 1 2 1 3 3 12 10.35 (3) 21 4 3 5 2 7 4 25 20.70 (4) 29 7 8 9 9 11 14 58 The frequency of chromosomal modifications in onion is directly correlated to the irradiation dose. (Graph 1). Graph 1 715 VII Radiation Physics & Protection Conference, 27-30 November 2004,Ismailia-Egypt Plate I - Chromosomal rearrangements induced by beta iradiation in Allium cepa Fig. 1. - Multiple chromatic telophase bridges derived from translocated chromosomes (arrow), Fig. 2. - Multiple micronuclei (arrow), Fig. 3. – Interphase nuclear heteropicnosis (arrow), Fig. 4. - Acentric fragments (a), chromosomal stickness (b), Fig. 5. - Dicentric chromosome (a), chromosomal gap (b), Fig. 6. - Ring chromosomes (a), acentric fragment (b) and a chain of chromosomes (c), Fig.7. - Double minutes (a) and a chain of translocated chromosomes, Fig. 8. – A chain of translocated chromosomes (a) and acentric ft(b) 716 VII Radiation Physics & Protection Conference, 27-30 November 2004,Ismailia-Egypt A large spectrum of chromosomal rearrangements was induced by beta irradiation, both in mice and in humans. The plate II (Figs. 1-6) reveals this broad spectrum of chromosomal rearrangements induced by beta irradiation in mice, including circularization of the telomeres and ring chromosomes (Figs. 1, 2). Robertsonian translocation generates biarmed chromosomes (Fig. 4), while telomere-to-telomere fusion generates dicentric chromosomes (Fig. 3). In some instances, the acentric fragments are abundant and are associated with translocations (Figs. 1, 6). The chromosomal breakage can generate gaps (Fig. 6) as well as deletions (Fig. 1) and acentric fragments. Centromere efilation is sometimes encountered (Fig. 5). Acentric fragments remain as separate structures or can be associated to the telomere of other chromosomes. (Table 2) Table 2 MICE CHROMOSOMAL MODIFICATION Gap (Gy) (Gy) Ring Ring Total Total doses Double minutes Acentric fragments Abnormal Irradiation metaphases Translocation chromosomes Control (0) 6 2 4 0 0 0 6 2.05 (1) 17 12 7 0 4 9 32 3.10 (2) 23 19 16 1 5 13 54 3.71 (3) 27 21 18 3 5 15 62 4.12 (4) 31 25 22 5 7 16 75 4.72 (5) 36 25 24 5 7 18 79 5.15 (6) 43 29 28 6 8 20 91 The frequency of chromosomal modifications in mice is directly correlated to the irradiation dose. (Graph 2). Graph 2 717 VII Radiation Physics & Protection Conference, 27-30 November 2004,Ismailia-Egypt Plate II - Chromosomal rearrangements induced by beta irradiation in Mus musculus domesticus Fig. 1. – Double minutes (a), Deletion (b), Circularization of telomeres (c), Acentric fragments (d). Fig. 2. - Ring chromosomes (arrow). Fig. 3. – A dicentric chromosome, as a result of telomere-to-telomere translocation with circularization (arrow). Fig. 4. - Robertsonian translocation (a), “Rosette” arrangement (b). Fig. 5. - Restructured chromosomes (a), Centromere efilation (b). Fig. 6. - Gap (a), Chain of acentric fragments (b), Multiple translocations (c). The spectrum of chromosomal abnormalities identified in humans (plate III, Figs.