Space radiation
Along with the long-term space exploration comes various potential health risks due to unique physical factors of the space environment.[1] Space radiation is one of the primary environmental hazards associated with space flight. Crew members are subjected to greater amounts of natural radiation in space than they receive on Earth, which can result in immediate and long-term risks. The three major sources of radiation in space are the trapped belt radiation, the galactic cosmic rays (GCRs), and the solar particle events (SPEs). Trapped belts of energetic particles, found in the Earth's magnetic field, consist predominantly of protons and electrons. GCRs consist of protons and heavy particles that originate outside the solar system. Solar flares are produced by solar magnetic storms and can last for hours or days. A solar particle event, which sometimes accompanies solar flares, may be the most potent space radiation hazard. Radiation exposure can lead to radiation sickness and has the potential to cause late effects (e.g., cancer in humans). Space radiation is different from gamma rays and neutrons in terms of energy absorption and ionization pattern. Although a significant amount of data on biological effects of gamma rays and neutrons has been obtained from atomic bomb survivors, there is very little human radioepidemiology data on bioeffects of high-energy charged particle radiation. A few studies with animals and cultured mammalian cells showed that energetic protons and heavy ions can effectively induce oncogenic cell transformation in vitro and tumors in vivo.[2,3.4] Thus, the basic mechanism(s) of radiation carcinogenesis remains to be elucidated. Even less known are the effects of charged particles on normal tissues. Limited experimental data indicate that heavy ions can be more effective than gamma rays in damaging normal tissues. We are using cultured epithelial cells as model systems to determine the short- and long-term responses of normal tissues to charged particles which may help to uncover the basic mechanism(s) of space radiation carcinogenesis.[5] The experimental data obtained from these studies will improve our understanding of the early and late effects of radiation on epithelia and provide insights into the mechanism of radiation carcinogenesis which are a major concern in radiation risk assessment for space flights.

Epithelial cells
The transport of ions and the ionic equilibrium across cell membranes are involved in all the physiological processes of living organisms. In mammals, the transport and asymmetric distribution of sodium across cell membranes is vital at all stages of development between conception and death. Epithelial cells produce ionic and solute translocation in several human organs (e.g., renal cells are responsible of urinary sodium reabsorption and extracellular fluid volume homeostasis). Epithelial cells have polarized plasma membrane surfaces with an "apical" domain (which faces the lumen) and a "basolateral" domain (which is closer to the blood vessels).[6] The two domains are demarcated by a ring of tight junctions. These specialized cell-cell junctions prevent the free movement of solutes and ions across the epithelium. Thus, epithelia separate two compartments with different composition in solute and ionic concentration. Apical and basolateral plasma membranes contain specific proteins which mediate the vectorial movement of solutes and ions across the epithelium. Maintenance of a polarized epithelial cell phenotype, a requirement for vectorial ion and solute transport, necessitates that i) newly synthesized proteins be post-translational processed and "sorted" in order to eventually reside in their specific cell surface domain; and ii) that the tight junctions be kept all the time.

Experimental project
Using epithelial cells, we are examining the early and late effects of radiation on the establishment and maintenance of tight junctions; and on the passive and active transports of solute and ions across epithelia. Since epithelia of different organs may have different sensitivity to radiation, we use cells from different tissues: a human mammary epithelial cell line transformed by ionizing radiation;[7,8] and a cell line from kidney tubules which is involved in urinary sodium reabsorption. In future experiments we will use retinal pigmented epithelial cells (which play an essential functional role in the eye). This last cell line is important since the eyes may be a very sensitive target for space radiation.

Normal cells, normal irradiated cells, and cells transformed by radiation are used. The electrical resistance is examined in confluent cell monolayers grown on collagen-coated polycarbonate filters. These filters are contained in small cups that totally separate the medium bathing the "apical" and "basolateral" cell surfaces. We measure the electrical resistance across the epithelium which is directly related to the establishment of the tight junctions.[6,9] Other physical and chemical techniques (like ion leakage and enzyme reactions) also will be used in future experiments to determine the establishment and maintenance of the tight junctions. The effect of radiation on the capacity to establish the tight junctions will be determined. Once the cells reach the maximal electrical resistance (confluence), the capacity of repolarization of the epithelium is tested. For this, confluent monolayers are incubated in calcium-free medium. This treatment releases the tight junctions and in this condition cells usually lose their polarization.[10] Normal and irradiated epithelium are tested for their capacity to re-establish a polarized epithelium. The re-establishment of the tight junctions are detected by the electrical resistance.

Measurement of solute and ionic transport across epithelia
In future experiments, ionic transport across the epithelium will be determined by measuring either the generation of a potential across the monolayer which is directly related to the amount of charges translocated using the technique of short circuit current or the transport of radioactive ions. The transport of solutes that are basic metabolites for the cells (carbohydrates, amino acids, etc.) will be determined with radioactive reagents. These studies will provide information about the effect of radiation on basic cellular processes that can be affected in the environment of outer space.

We will also determine whether radiation-induced transformation alters the mechanism of protein sorting. Immunofluorescence with antibodies that recognize membrane proteins will be used to determine the location of the antigens in monolayers of transformed cells.

References
1D. E. Robbins and T. C. Yang. "Radiation and Radiobiology," in Space Physiology and Medicine. Ed. A. E. Nicogossian, C. L. Huntoon, and S. L. Pool. 1994: Lea & Febiger, Philadelphia, PA. Chp. 9.
2C. H. Yang, L. M. Craise, M. Durante, and M. Mei. "Heavy-Ion Induced Genetic Changes and Evolution Processes," Adv. Space Res. 14 (1994): 373-82.
3T. C. Yang, K. A. George, M. Mei, M. Durante, and L. M. Craise. "Radiogenic Cell Transformation and Carcinogenesis," Science Issue ASGSB Bulletin 8 (1995): 106-12.
4T. C. Yang, M. Mei, K. A. George, and L. M. Criase. "DNA Damage and Repair in Oncogenic Transformation by Heavy Ion Radiation," Adv. Space Res. 18 (1996a): 149-58.
5T. C. Yang and L. M. Craise. "Development of Human Epithelial Cell Systems for Radiation Risk Assessment," Adv. Space Res. 14 (1994): 115-20.
6J. S. Handler. "Use of Cultured Epithelia to Study Transport and its Regulation," J. Exp. Biol. 106 (1983): 55-69.
7M. Durante, G. F. Grossi, and T. C. Yang. "Radiation-Induced Chromosomal Instability in Human Mammary Epithelial Cells," Adv. Space Res. 18 (1996): 99-108.
8T. C.Yang, K. A. George, A. Tavakoli, L. M. Craise, and M. Durante. "Radiogenic Transformation of Human Mammary Epithelial Cells in vitro," Radiation Oncology Investigation 3 (1996b): 412-19.
9C. 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.
10R. G. Contreras, G. Avila, C. Gurierrez, J. J. Bolivar, L. Gonzalez-Mariscal, A. Darzon, G. Beaty, E. Rodriguez-Boulan and M. Cereijido. "Repolarization of Na,K Pumps During Establishment of Epithelial Monolayers," Am. J. Physiol 257 (1989): C896-905.