Space Radiation Environment and Its
Biological Effects

Carlos H. Pedemonte, Ph.D., Assistant Professor, UH
Tracy C. Yang, Ph.D., JSC
Penny K. Riggs, Ph.D., Post-Doctoral Fellow, UH

SPACE RADIATION is a significant environmental hazard associated with space flight. Astronaut crews are subjected to greater amounts of natural radiation in space than they receive on Earth, a situation which imposes immediate and long-term health risks. Three major sources of radiation encountered in space include trapped belt radiation, galactic cosmic rays (GCRs), and solar particle events (SPEs). Trapped belts of energetic particles found in the Earth's magnetic field predominately consist of protons and electrons. Protons and heavy ion particles originating outside the solar system make up the GCRs. During solar flares produced by solar magnetic storms, SPEs may occur, producing protons and generating a potent space radiation hazard. Although data exist for acute human exposure to gamma rays, the physiological consequence of low-level exposures to the high-energy charged-particle radiation encountered in space is not well understood. Previous studies demonstrated oncogenic cell transformation induced by exposure to energetic protons and heavy ions,[1] but the mechanisms by which this transformation to a cancerous state occurs remain unclear.

Dr. Penny K. Riggs, Post-Doctoral Fellow, UH, (l.) with Dr. Carlos H. Pedemonte, in the UH College of Pharmacy, study health risks to humans in space.

Objectives
A goal of space radiation biology includes development of methods for prevention of potential radiation-induced cancers or other disorders during and after long-term space flights. As a step toward that goal, we are utilizing epithelial cells as model systems to determine the response of "normal" cells to charged particles in an attempt to elucidate mechanisms associated with radiation carcinogenesis. Ion transport and ionic equilibrium across cell membranes are physiological processes essential in all living organisms. Mammalian epithelial cells possess polarized plasma membrane surfaces, and cell-to-cell tight junctions prevent movement of solutes and ions across the epithelium. Transepithelial electrical resistance (TER) is a measure of the movement of ions across the paracellular pathway. Measurement of TER across cells grown on permeable membranes can provide an indirect assessment of tight junction establishment and stability.[2] Because the epithelium separates cellular compartments with very different fluid composition, maintenance of its stability and electrical resistance is critical for essential physiological processes. Therefore, changes in TER may represent an early expression of cell damage. The object of this work was to determine whether TER measurements in cell culture monolayers can be used as a model system for detecting cell damage produced by exposure to radiation and for testing potential chemoprotective agents.

Methodology
Madin-Darby cells (MDCK), a canine kidney urinary epithelial cell line,[3] were maintained in culture. As a result of characteristics of the irradiation chamber and the necessity to maintain sterile conditions, irradiation was performed on cells in suspension. Cultures were grown in plastic flasks, then resuspended in culture medium. A fraction of the cells was reserved as a non-irradiated control, and the remaining cells were exposed to gamma or heavy ion (C 290 MeV/u, 13.25 KeV/m; or Ne 400 MeV/u, 30.96 KeV/m) irradiation at doses of 5 to 25 Gy. Gamma irradiation was performed in Houston, Texas, at the Baylor College of Medicine in a Gammacell 1000; Cs¹³⁷). Heavy ion exposure was accomplished at the National Institute of Radiological Sciences (NIRS) heavy ion medical accelerator (HIMAC) in Chiba, Japan. After irradiation, cells were grown as monolayers on polycarbonate filters in six-well tissue culture plates. Six filters were plated for each radiation dose, and an excess of cells was applied so that the entire surface of the filters would be covered by confluent cells shortly after plating. Cell culture medium was applied to both sides of the filter representing the apical and basolateral cell surfaces, and changed every two days. Electrical resistance across cell membranes was measured daily over a period of up to ten days with chopstick-style Ag⁺/AgCl₂ electrodes attached to a portable ohmmeter. Additional cell cultures were irradiated, and samples were preserved for future studies to correlate gene expression with membrane behavior.

Results
MDCK cells in suspension were exposed to gamma or heavy ion (Carbon 290 MeV/u, 13.25 KeV/um or Neon 400 MeV/u, 30.96 KeV/um) irradiation. After irradiation, cells were seeded onto filters and the development of TER was measured daily. While normal cells have a stepwise increase in TER up to eight days, Figs. 1(a)-1(c) illustrate that the development of TER is significantly reduced in irradiated cells as compared to controls. Mean TER was computed for each treatment and plotted against days in culture. Beyond the third day after seeding, TER for cells exposed to the three types of radiation were significantly different from controls (p < 0.0001). In all cases, irradiated cells displayed retarded development of TER. The level of TER reduction was dose-dependent, but the 10 Gy and 25 Gy groups did not differ from each other.

Figures 2(a)-2(c) indicate the level of TER reduction as a percentage of the control TER at different doses of radiation. Generally, for the first few days, carbon-irradiated cells maintain slightly higher TER levels than either neon- or gamma-irradiated cells. Beyond day four, however, the cellular response to irradiation appear to be similar despite differences in radiation quality.

A possible cause of the reduced TER observed in irradiated cells is that a few cells die but remain in the monolayer with cells that are alive. As cells die, tight junctions would be loosened, but this hypothesis does not explain our results. Trypan blue vital dye was used to stain MDCK cells that had been exposed to 25 Gy gamma rays 10 days previously. Although TER levels were less than half the TER of the control cells, these treated cells remained confluent and were able to exclude dye. Therefore, the reduced TER observed in irradiated samples does not appear to result from dead cells intercalated between viable cells.

Left. Dr. Carlos H. Pedemonte conducts research on Astronaut crews subjected to greater amounts of radiation in space than on Earth.

Discussion
Exposure to heavy ion radiation caused a significant dose-dependent reduction in TER of renal epithelial cells. The decreased TER is not simply due to cells killed by radiation damage. First, dead cells cannot establish tight junctions with neighbor cells and, therefore, will not be included or will be immediately eliminated from the monolayer. Second, dead cells intercalated in the monolayer should produce a TER near zero and not close to that of the control cells we determined. Third, we did not observe any non-viable cells in monolayers of cells exposed to 25 Gy gamma irradiation and stained with trypan blue vital dye. Therefore, our results suggest that the ability of epithelial cells to establish and maintain tight junctions, i.e., normal physiological function, was impaired after irradiation. This conclusion supports the observation that tight junctions were "leaky" in intestinal epithelial cells from adult rats exposed to gamma rays.[4,5] Walker and colleagues[5] observed that aseptic endotoxemia may be a consequence of irradiation injury at dose levels that do not denude the intestine of epithelial cells. Under normal conditions, the epithelium should not be permeable to large-molecular-weight substances such as bacterial endotoxins. Porvaznik4 showed by electron microscopy that tight junctional structures were disrupted after irradiation. The author suggested that tight junction fragments were produced as a result of radiation damage and removed by phagocytosis since he observed these fragments in cytoplasmic vesicles between days three and seven after irradiation. Eventually, the "leaky" ileal tight junctions returned to normal after seven days. This return-to-normal was not observed in the cell culture system we tested and may be attributed to the fact that some mechanisms of cell repair (e.g. phagocytosis) may not be present in vitro. Our observations, along with those of Porvaznik, suggest that tight junctions are a sensitive target for radiation. Part of the pathological condition known as radiation injury may be associated with the disruption of tight junctions since these may provide a permissive route that allows the penetration of microbial agents and toxins normally retained in the apical side of the epithelia.

We are unsure of the origin of the reduced TER caused by radiation. Haimovitz-Friedman and colleagues[6] demonstrated that ionizing radiation can act on cellular membranes to initiate apoptosis (self-induced killing of damaged cells) in bovine aortic endothelial cells. These authors suggest that ionizing radiation acts at the membrane to stimulate a cascade of events involving the hydrolysis of sphingomyelin, mimicking the effects of tumor necrosis factor (TNF-a) which is known to induce apoptosis.[7] Indeed, it has been described that TNF-a may produce apoptosis in LLC-PK1 renal epithelia cells[8] and cause decreases in TER.[9] Thus, it is possible that the radiation-dependent reduction in TER, which we observed, is related to the occurrence of apoptosis. Further experiments will be required to test this hypothesis.

Ongoing and Planned Experiments
The present studies illustrate that determination of TER provides a useful model for testing the effects of space radiation. This model is an in vitro system that uses live cells to determine physiological parameter essential for living organisms. As our results indicate, changes in TER can be detected quickly after irradiation. By this method, we can study subtle changes along a potential pathway to carcinogenesis. We also intend to use this system to test the efficacy of agents used for chemoprevention of radiation damage. Reagents known to counteract cellular damage and stimulate immune response including free-radical scavengers, electrophile scavengers, peroxidase inhibitors, and other agents such as antioxidants found in green tea and other foods, can be combined as a "chemoprevention cocktail" and tested as a radioprotective agent.[10] Previous attempts at radioprotection in other laboratories have not had sufficient success because of low efficacy or high toxicity or because of the requirement of parenteral administration. In preliminary experiments, we tested the chemoprevention capacity of a cocktail of reagents with multiple mechanisms of action. The rationale behind the composition cocktail holds that the use of a complex of agents may be more effective than a single agent alone would allow administration of lower doses, thus lessening toxicity. Early data (not shown) indicate that treatment with the cocktail provided some protection to samples irradiated with accelerated carbon ions. These results were encouraging, thus additional experiments are underway. We will be testing individual components, as well as varying compositions, concentration, and treatment durations of the chemoprevention cocktail.

We are also accumulating samples of RNA from irradiated cells. These samples are being tested to determine which genes become transcriptionally active, or get "turned on," after irradiation, and we have accumulated data demonstrating differential expression of transcripts. We hope to correlate changes in levels of gene expression with the changes in TER as well as with the administration of chemoprevention cocktails. Observations of aberrant changes in gene expression may provide insight into the physiological changes associated with carcinogenesis.

Other scheduled experiments involve studying the interaction between microgravity and radiation exposure. Preliminary data indicate that chemicals which mimic the effects of microgravity by inducing changes in membrane ordering can also cause changes in TER levels. Future testing will determine whether microgravity and radiation have a synergistic effect on TER across epithelial membranes.

Collaborators
Kerry George of Krug Life Sciences has provided invaluable technical assistance at JSC. Dr. Yoshiya Furusawa and the National Institute of Radiological Sciences (NIRS) of Japan kindly provided the opportunity to conduct experiments at the heavy ion medical accelerator at Chiba (HIMAC). Dr. Jeff Jones, JSC Flight Medicine Program and USRA is providing the chemoprevention cocktail and collaborating in those studies. Dr. Mark Clarke of the JSC Muscle Research Lab provided reagents for the simulated microgravity work. This ISSO/NASA project began as an interaction between the University of Houston and the Radiation Biophysics Laboratory at JSC. However, interaction and collaborations associated with this project now include Krug Life Sciences, NRC postdoctoral scientists, NIRS (Japan), UTMB, Baylor University, Texas A&M, and several additional laboratories at the Johnson Space Center.

Dr. Tracy C. Yang conducts research in the field of space radiation biology. In a protected environment, Dr. Yang seeks to develop methods for the prevention of potential radiation-induced cancer during and after long-term space flights. Epithelial cells are utilized to help determine the response of "normal" cells to charged particles. Transepithelial electrical resistance (TER) is a measure of the movement of ions across paracellular pathways.

References
[1]T. C. Yang, K. A. George, A. Tavakoli, L. Craise, and M. Durante. "Radiogenic Transformation of Human Mammary Epithelial Cells in vitro," Radiat. Oncol. Invest. 3 (1996): 412-19.
[2]C. H. Pedemonte. "Inhibition of Na-Pump Expression by Impairment of Protein Glycosylation Is Independent of the Reduced Sodium Entry Into the Cell," J. Membrane Biol. 147 (1995): 223-31.
[3]C. R. Gaush, W. L. Hard, and T. P. Smith. "Characterization of an Established Line of Canine Kidney Cells (MDCK)," Proc., Soc. Exp. Biol. Med. 122 (1996): 931-35.
[4]M. Porvaznik. "Tight Junction Disruption and Recovery after Sublethal Gamma Irradiation," Radiat. Res. 78 (1996): 233-50.
[5]R. I. Walker, G. D. Ledney, and C. B. Galley. "Aseptic Endotoxemia in Radiation Injury and Graft-vs-Host Disease," Radiat. Res. 62 (1975): 242-49.
[6]A. Haimovitz-Friedman, C.- C. Kan, D. Ehleiter, R. S. Persaud, M. McLoughlin, Z. Fuks, and R. N Kolesnick. "Ionizing Radiation Acts on Cellular Membranes to Generate Ceramide and Initiate Apoptosis," J. Exp. Med. 180 (1996): 525-35.
[7]D. W. Jarvis, R. N. Lolesnick, F. A. Fornari, R. S. Traylor, D. A. Gewritz, and S. A. Grant. "Induction of Apoptotic DNA Degradation and Cell Death by Activation of the Sphingomyelin Pathway," Proc., Nat'l Acad. Sci. USA 91 (1974): 73.
[8]A. Peralta Soler, J. M. Mullin, K. A. Knudsen, and C. W. Marano. "Tissue Remodeling During Tumor Necrosis Factor-Induced Apoptosis in LLC-PK1 Renal Epithelial Cells," Am. J. Physiol. 270 (1996) (Renal Fluid Electrolyte Physiol. 39): F869-F879.
[9]C. W. Marano, K. V. Laughlin, L. M. Russo, A. Peralta Soler, and J. M. Mullin. "Modulation of Tumor Necrosis Factor-induced Increase in Renal (LLC-PK1) Transepithelial Resistance," J. Cell Physiol. 157 (1996): 519-27.
[10]M. Stanford and J. A. Jones. "Space Radiation Concerns for Manned Exploration," Acta Astronomica (1997). (Submitted for publication.)

Contents
ISSO -- Institute for Space Systems Operations
1996-1997 Annual Report