1 Alexander Vaglenov*, Boris Fedorenko**RBE AND GENETIC SUSCEPTIBILITY OF MOUSE AND RAT SPERMATOGONIA TO PROTONS, HEAVY CHARGED PARTICLES AND NEUTRONS Alexander Vaglenov*, Boris Fedorenko** The University of Findlay, College of Pharmacy, OH, USA* National Centre of Radiobiology and Radiation Protection, Sofia, Bulgaria* and Institute for Biomedical Problems, Moscow, Russian Federation** Results cont. Introduction Currently, on mission to the moon, Space Stations, or Mars a high priority is understanding the risk of the late effects from exposure to SPE-solar particle events and GCR-galactic cosmic radiation, such as cataracts, cancer, neurological disorders, degenerative tissue and hereditary effects (Cardis et al., 2007; Cavallo et al., 2002; Cucinotta et al., 2006, 2008, 2014; Durante et al., 2008; Fedorenko et al., 2000, 2010, George et al., 2001, 2003; Hall et al., 2006; Langner et al., 2004; Nicholas et al., 2003; Picco et al., 1999, 2000; Vaglenov et al., 2007). There are three groups of individuals heavily exposed to high-LET radiation during their careers, warranting consideration for genetic risk assessment: First, on intercontinental flights, at altitudes around 10,000–12,000 meters, the estimated mean cumulative exposure for civil aviation air crew is 3 mSv per year, with a range from 1 to 10 mSv per year. On a second place are astronauts in International Space Station with a range from 44 to 105 mSv. On a Third place are long-term interplanetary mission, such as travel to Moon, and Mars or into deeper space, it is expected that astronaut crews will accumulate doses of radiation of around 1 Sv or higher. Most of our knowledge of the genetic effects of exposure to heavy charged particles comes from either accelerator-based experiments or radiobiological studies conducted directly in space. The latter have the advantage of including interaction of all other space environmental factors, but are very expensive. Material and Methods cont. Conclusions Table 1. Irradiation type, dose/rate, dose-range and animals Relative to 60Co gamma-rays, RBE values are as follows for mouse spermatogonia: 0.9 for 50 MeV protons; 1.3 for 9 GeV protons; 0.7 for 4 GeV helium ions; and 1.3 for 4 GeV carbon ions. For rat spermatogonia, values were: 1.7 for 9 GeV protons and 1.3 for helium ions The genetic susceptibility of the rat to the corpuscular radiations investigated exceeded the susceptibility observed in the mouse. Taking into consideration the evidence from both mammalian species, we suggest that the rat is more susceptible than the mouse to protons and HZE particles. Species Radiation Dose / rate Gy / min Dose-range Gy Numbers RBE Animals Cells Mouse Protons 50MeV 0.4–3.0 (1.26 keV/µm) 21 3,980 0.9 Protons 9GeV 1.2–2.0 (0.23 keV/µm) 33 6,800 1.3 Helium ions 4GeV 0.5–1.5 (8.8 keV/µm) 0.5 – 4.0 27 5,323 0.8 Carbon Ions 4GeV 0.2–0.6 (7.6 keV/µm) 28 5,600 Neutrons 1.5MeV 0.25 (40.0 keV/µm) 0.15 – 1.5 50 10,000 6.6 Gamma-rays 0.36 (0.25 keV/µm) 0.15 – 4.0 52 10,400 1.0 Control 10 2,000 Rat Protons 9 GeV (0.23 keV/µm) 6,477 1.7 (8.8 keV/µm) 0.5 – 3.0 20 3,923 4,000 8 1,600 References: Cardis E et al. The 15-country collaborative study of cancer risk among radiation workers in the nuclear industry: Estimates of radiation-related cancer risks. Radiat. Res., 167, , 2007. Cucinotta FA. Space radiation risk for astronauts on multiple International Space Station missions. PLOS ONE, 9 (4), 1-14, 2014. Cucinotta FA, Durante M. Cancer risk from exposure to galactic cosmic rays: implications for space exploration by human beings. Lancet Oncol., 7, , 2006. Cucinotta FA, Kim MY, et al. Physical and biological dosimetry analysis from International Space Station astronauts. Radiat Res, 170, , 2008. Durante M., Cucinotta FA. Heavy ion carcinogenesis and human space exploration. Nat Rev Canc, 8, , 2008. Dyban A. Preparation technique for meiotic chromosome slides from mammalian testes. Tsitology 12, , 1970 (in Russian) Evans EP, Breckon, G, Ford CE. An air drying method for meiotic preparation from mammalian testes. Cytogenetics 3, , 1964. Cavallo D, Marinaccio A, Perniconi B, et al. Chromosomal aberrations in long-haul air crew members. Mutat Res, 513, Fedorenko BS, Shevchenko VA, Snigiryova GP et al. Cytogenetic studies on cosmonauts blood lymphocytes after long-term manned space flights. Radiat Biol Radioecology, 40, , (in Russian) Fedorenko BS, Vaglenov A, Abrosimova A. Cytological and cytogenetic damages of spermatonial cells in mice in acute and late periods after irradiation by protons, helium ions, and gamma-rays. Radiat Biol Radiaoecology, 50, , 2010. George K et al., Chromosome aberrations in the blood lymphocytes of astronauts after spaceflight. Radiat Res, 156, , 2001. George K, Durante M, Wu H et al. Biological effectiveness of accelerated particles for the induction of chromosome damage measured in metaphase and interphase human lymphocytes. Radiat Res, 160, , 2003. Hall EJ, The relative biological effect6iveness of densely ionizing heavy-ion radiation for inducing ocular cataracts in wild type versus mice heterozygous for the ATM gene. Radiat Environ Biophys, 45, , 2006. Langner I, Blettner M, Gundestrupt M. et al. Cosmic radiation and cancer mortality among airline pilots: results from European cohort study (ESCAPE). Radiat Environ Biophys 42, Nicholas JS, Butler GC, Davis S, et al. Stable chromosome aberrations and ionizing radiation in airline pilots. Aviat. Space Environ Med 74, , 2003. Picco SJ, Luca LC, McIntyre C et al. Chromosomal damage in air crew members of international flights. A preliminary report. Genet Mol Biol, 23, , 2000. Vaglenov et al, RBE and genetic susceptibility of mouse and rat spermatogonial stem cells to protons, heavy charged particles and 1.5 MeV neutrons. Advance in Space Res. 39, , 2007. Fig. 1. Diakinesis-metaphase I Spermatocytes: Objectives The aim of this study is to establish the RBE-relative biological effectiveness of mouse and rat spermatogonial stem cells to protons, heavy charged particles (HZE) and neutrons by using RT-reciprocal translocation induction. Reciprocal translocations (RT) in mammalian spermatogonia are a well-established, classical endpoint in radiobiology, and assessing RT in mammalian spermatogonia is considered to be a reliable approach for predicting genetic radiation risk. A C RIV CIV B D Material and Methods Animals: All adult males (CBA · C57Bl/6J) F1 mice and Wistar rats were sacrificed six months after irradiation, and their testes were removed and used for cytogenetic examination. Animals were kept under standard housing conditions and fed commercial food pellets. Irradiation: Using the synchrophasotron at the Joint Institute of Nuclear Research (JINR) in Dubna, Russia, we irradiated animals with HZE particles such as helium ions, carbon ions, and deuterons of high energies. Mice were irradiated with neutrons of 1.5 MeV on JINR’s Fast Impulse Reactor in Dubna. The irradiation was performed at a dose–rate of 0.25 Gy/min. A gamma unit of 60Co was used to expose animals to gamma-rays at a dose–rate of 0.36 Gy/min (see Tale 1). Slide preparation: Six months after irradiation, genetic damage was assessed by recording yields of reciprocal translocations induced in mouse and rat spermatogonia, the only endpoint parameter for which limited data have been obtained after induction in irradiated human spermatogonia. Slides were prepared as described by Evans et al., 1964 for mice, as well as Dyban (1970) for rats. From each animal, 200 metaphase figures of spermatocytes in diakinesis-metaphase I were examined. At diakinesis-metaphase I, 20 bivalents for mice and 21 for rats are normally formed. At this stage of spermatogenesis, translocations between nonhomologous chromosomes appear as multivalents (see Fig 1A, B, C, and D). Two independent scorers analyzed coded slides at 1200-fold magnification, checking for multivalent configurations indicative of reciprocal translocations. A - mouse metaphase with normal bivalents set, n = 20 B - mouse metaphase with chain of four bivalents, 18 + CIV C - rat metaphase with normal bivalents set, n = 21 D - rat metaphase with 2 rings of four bivalents, RIV Results RT yields measured after mouse and rat spermatogonial irradiation with protons, heavy charged particles and neutrons fit the linear model of the dose–response relationship. The spontaneous RT frequency in mouse spermatogonia is very low – one RT was found after examination of 2000 cells from 10 control mice. The ratios of the slopes of the dose-response curves of mice spermatogonia exposed to protons and different high energy and charge particles (HZE) compared to the slopes of exposure to gamma-rays determined the RBE. RBE for 50 MeV protons is 0.9, 1.3 for 9 GeV protons, 0.7 for 4 GeV helium ions, and 1.3 for 4 GeV carbon ions (Fig 2A and Table 1). The RBE of 1.5 MeV neutrons is about 6.6 (Fig. 2B and Table 1). Analysis of 1600 spermatociyes at diakinesis-metaphase I from 8 control rats did not show reciprocal translocations. Fig. 1C shows that protons and helium ions induced more genetic damage than standard gamma-irradiation. The RBE for 9 GeV protons is 1.7, and for 4 GeV helium ions is 1.3 (Fig. 2C and Table 1). Fig. 2. Dose dependence of RT after acute irradiation of mice and rats spermatogonia with protons and high energy and charge particles: a) mouse spermatogonia irradiated with protons and HZE particles; b) mouse spermatogonia irradiated with 1.5 MeV neutrons and c) rat spermatogonia irradiated with 9 GeV protons and 4 GeV helium ions. 62nd Annual International Meeting of Radiation Research Society, October , 2016, Waikoloa Village-Big Island, Hawaii