SPACE RADIATION AND MICROGRAVITY ARE TWO MAJOR ENVIRONMENTAL STRESSORS FOR HUMAN IN SPACE TRAVEL. ONE OF THE FUNDAMENTAL QUESTIONS IN SPACE BIOLOGY RESEARCH IS WHETHER THE COMBINED EFFECTS OF MICROGRAVITY AND EXPOSURE TO COSMIC RADIATION ARE SYNERGISTIC. WHILE STUDIES ADDRESSING THIS QUESTION HAVE BEEN CARRIED OUT FOR HALF A CENTURY IN SPACE OR USING SIMULATED MICROGRAVITY ON THE GROUND THE REPORTED RESULTS ARE CONFLICTING. ALTHOUGH THE REASON FOR THE VARIATION IN RESULTS IS NOT KNOWN IT IS POSSIBLE THAT IT MAY BE DUE TO THE DIVERSITY OF BIOLOGICAL SYSTEMS USED BUT MORE IMPORTANTLY TO THE EXPERIMENTAL DESIGNS AND HARDWARE USED IN THESE STUDIES. FOR THE ASSESSMENT AND MANAGEMENT OF HUMAN HEALTH RISKS IN FUTURE MOON AND MARS MISSIONS IT IS NECESSARY TO OBTAIN MORE BASIC DATA ON THE MOLECULAR AND CELLULAR RESPONSES TO COMBINED EFFECTS OF RADIATION AND MICROGRAVITY. TO ESTABLISH A FIRM BASELINE DATABASE WE PROPOSE TO UNDERTAKE A SYSTEMATIC STUDY ON CULTURED MAMMALIAN CELLS' RESPONSES TO THE SIMULTANEOUS INSULT OF RADIATION AND MICROGRAVITY (BOTH IMMEDIATE AND LONG TERM) TO ELUCIDATE THE MOLECULAR SIGNALING PATHWAYS THAT LEAD TO THESE BIOLOGICAL EFFECTS. THE RESULTS OF THE STUDY WILL PROVIDE CELLULAR AND MOLECULAR BIOLOGICAL BASES FOR THE ASSESSMENT AND MANAGEMENT OF HUMAN HEALTH RISKS IN SPACE. RECENTLY DR. TAKAHASHI CO-I OF THIS PROPOSAL HAS DEVELOPED MICROGRAVITY-IRRADIATION SYSTEMS CONSISTING OF A 3D CLINOSTAT SYNCHRONIZED TO THE CARBON-ION OR X-RAY IRRADIATION SYSTEMS. OUR NEW EXPERIMENTAL SETUP ALLOWS US TO AVOID STOPPING CLINOSTAT ROTATION DURING IRRADIATION WHICH WAS REQUIRED IN ALL OTHER PREVIOUS EXPERIMENTS. GUNMA UNIVERSITY HEAVY ION MEDICAL CENTER IS THE ONLY FACILITY IN THE WORLD WHERE WE CAN EXPOSE SAMPLES TO HIGH-LINEAR ENERGY TRANSFER (LET) IRRADIATION AS WELL AS LOW-LET IRRADIATION UNDER THE SIMULATED MICROGRAVITY CONDITION (I.E. WITHOUT INTERRUPTING CLINOSTAT ROTATION). OUR PRELIMINARY DATA OBTAINED FROM THE USE OF THIS NEW DEVICE ON GENE EXPRESSION IN HUMAN FIBROBLASTS SHOW THAT SPLICING CYCLERELATED GENES AND CELL CYCLE RELATED GENES ARE SIGNIFICANTLY UP-REGULATED AND S-PHASE DNA REPLICATION AND DNA REPAIR-RELATED GENES WERE DOWN-REGULATED WITH C-ION IRRADIATION UNDER SIMULATED MICROGRAVITY. IN THIS PROPOSAL WE WILL INVESTIGATE 3 DIFFERENT ENDPOINTS FROM EARLY TO LATE RESPONSES IN 2 HUMAN CELL LINES USING OUR NEW DEVISES TO STUDY COMBINED EFFECTS OF MICROGRAVITY AND SPACE RADIATION. HUMAN FIBROBLASTS AND EPITHELIAL CELLS WILL BE EXPOSED TO X-RAYS AND C-IONS UNDER THE SIMULATED MICROGRAVITY CONDITION (ROTATED WITH 3-D CLINOSTAT). CONTROL CELLS WILL BE IRRADIATED IN 1G ENVIRONMENT (WITH THE STATIC STAGE). WE WILL INVESTIGATE THE EXTENT OF EXPRESSION OF SPECIFIC PROTEINS AND OF THE POST-TRANSLATIONAL MODIFICATION STATES OF SIGNALING PROTEINS (AIM 1) GENE EXPRESSIONS AND THE PATHWAYS INVOLVED (AIM 2) AND THE EXTENT OF CHROMOSOME ABERRATIONS (AIM 3) CAUSED BY THE COMBINED EFFECTS OF SIMULATED MICROGRAVITY AND RADIATION. TO INVESTIGATE FROM THE EARLY TO LATE ENDPOINTS IN THE SAME CELL TYPES WILL PROVIDE CELLULAR AND MOLECULAR BIOLOGICAL DATA THAT ARE NEEDED TO UNDERSTAND THE IMPACT OF COMBINED EFFECTS OF SIMULATED MICROGRAVITY AND SPACE RADIATION ON HUMAN HEALTH. ONE OF THE SELECTED ENDPOINTS IS CHROMOSOME ABERRATIONS WHICH IS A WELL-ESTABLISHED BIOMARKER FOR CANCER RISK AND HAS BEEN USED BY NASA FOR THE RISK ASSESSMENT OF ASTRONAUTS. STUDYING THIS ENDPOINT ALLOWS US TO COMPARE OUR RESULTS TO THE ASTRONAUTS' DATA AFTER THEIR ISS MISSIONS. COMPLETION OF THIS PROPOSAL WILL ALLOW US TO DETERMINE HOW THE COMBINATION OF MICROGRAVITY AND RADIATION WILL AFFECT THE TRANSCRIPTOMIC METABOLOMIC AND PROTEOMIC STATES OF CELLS AS WELL AS HERITABLE CHANGES IN DNA. THESE FINDING WILL ALLOW US TO HELP DEVELOP THE COUNTERMEASURES FOR THE FUTURE SPACE MISSIONS.