Biological effects of low dose irradiation of a vertebrate embryo, Japanese medaka (Oryzias latipes), with a high-energy secondary neutron spectrum

W. W. Kuhne,¹ B. B. Gersey,² R. Wilkins,² H. Wu,³ S. A. Wender,⁴ W. S. Dynan,¹

¹Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, Georgia

²Center for Applied Radiation Research, Prairie View A & M University, Prairie View, Texas.

³NASA Johnson Space Center, Houston, Texas.

⁴Los Alamos Neutron Science Center, Los Alamos National Laboratory, Los Alamos, New Mexico

Galactic cosmic, solar particle, and trapped particle radiation produce secondary radiation in the form of high-energy neutrons by interacting with the atmosphere, spacecraft structure, and planetary surfaces. These high-energy neutrons contribute to the absorbed dose and dose equivalent received by crew members and passengers during commercial aviation travel and astronauts in space missions. This study was conducted utilizing the broad-spectrum, high-energy neutron beam delivered at the ICE House 30L facility at the Los Alamos Neutron Science Center (LANSCE), Los Alamos, New Mexico. The neutron energy spectrum delivered at the ICE House 30L facility is similar in shape to the energy spectra of secondary neutrons found both in spacecraft and in the upper atmosphere.

In this work, we investigated the biological damage expressed in developing embryos of the Japanese medaka fish (Oryzias latipes) after these embryos were exposed to low doses of neutron radiation at the ICE House 30L facility. As vertebrates, medaka share with humans a large complement of vertebrate-specific organs of radiobiological interest (brain, spinal cord, vasculature, digestive, excretory, and hematopoietic systems). Specifically, we quantified the number of apoptotic cells arising in medaka embryos 30 hours after exposure by use of a whole mount TUNEL assay. Embryos were stained with rhodamine-labeled anti-digoxigenin FAB fragments (Roche Applied Science, Indianapolis) and cleared with benzyl amino benzoate immediately prior to imaging to aid in uniform detection of staining signals through the depth of the embryo. Confocal images were collected using a Zeiss LSM 510 confocal laser scanning microscope. The rhodamine fluorphore was excited using 543 nm He:Ne laser illumination and confocal images were collected using a 20X objective with a 5 µm step size, resulting in approximately 100 slices per embryo. From the Z stack images at different focal planes, we created 3D renderings of each embryo. TUNEL-positive cells were counted using Volocity 3D imaging software (Improvision, Lexington, Massachusetts) with image acquisition, volume visualization and object classification modules.

The purpose of this experiment was to investigate the biological damage using an in vivo model system and to establish an RBE value in comparison to low-linear energy transfer(LET) radiation. Medaka embryos, Stage 25-27, were exposed to high energy neutrons at the ICE House 30L facility. Embryos received total absorbed doses ranging from 0.1 to 0.5 Gy at a doserate of 0.015 Gy h^-1. Dosimetry was performed using both a well-characterized neutron Time Of Flight (TOF) system, and a Tissue Equivalent Proportional Counter (TEPC). Preliminary results of apoptosis measured in a defined portion of the tail indicate statistically significant differences in TUNEL-positive cells measured at doses of 0.2, 0.3, and 0.5 as compared to embryos receiving the 0 Gy dose. Medaka embryos exposed to 137Cesium gamma-rays to doses of 0, 0.15, 0.5, 1.5, 5, and 15 Gy, at a dose-rate of 1.87 Gy h^-1 showed significant differences in levels of TUNEL-positive cells starting at doses of 1.5 Gy. Preliminary analysis of survival of irradiated embryos at 10-days post-hatch and apoptosis levels indicate an RBE ~30. Additional analysis is being conducted to measure TUNEL-positive cells in the head and eyes of the embryo and final results will be presented.

Over the past 20 years studies concerning the response of the medaka to ionizing radiation have been a subject of intense study and have resulted in a significant body of literature. However, only a few papers have resulted from studies that specifically investigated the response of the medaka to high-LET radiation, and to our knowledge this would be the first medaka study using a high-energy neutron beam.

Supported by the Low Dose Radiation Research Program, Grant No. DOEFG02-03ERG3649

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