. - --- . * . .! : . 7 * i I OFI ORNL P 1734 .. ... .. . ol * * · mi 17 11. : . : : 1. i - 45 - :.-: ' - i. - 1 11:25 | 1.4 1.1.6 MICROCOPY RESOLUTION TEST CHART NATIONAL BUREAU OF STANDARDS - 1963 .. 2 . ... : more ....* . .. MON 180 orrr -p-i7 34 Conf-651042-1 Manned Space Flight Experiments Symposium Gemini Missions 3 and 4 Washington, D.C., October 19, 1965 E زنانه قد ناخن LEGAL NOTICE This report was prepared as an account of Government sponsored work. Neither the United Statos, dor the Commission, nor any person acung on behalf of the Commission: A. Makes any warranty or representation, expressed or implied, with respect to the accu- racy, completeness, or usefulness of the Information contained in the report, or that the use of any information, apparatua, method, or process disclosed in this report may aot infringe privately owned rights; or B. Assumos any liabilities with rospect to the use of, or for damages resulung from the use of any information, apparatus, method, or process disclosed in this report. As used in the above, "person acting co behalf of the Commission” includes any em- ployee or contractor of the Commission, or employee of such contractor, to the exten' that such employoo or contractor of the Commission, or employee of such contractor preparos, dissominates, or provides access to, any information pursuant to his omployment or contract with the Commission, or his employment with such cortractor. EXPERIMENT S4, RADIATION OF BLOOD" Michael A Bender, P. Carolyn Gooch, and Sohei Kondo? ---- - Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee -..-on. nin KRUUSTED TO AMPOUNCUENT IN MUCILAR SCIENCE ABSTRACTS .. 'Research carried out at ORNL under NASA Order Number R-104 Task 4. "Prosent address: Dept. of Fundamental Radiology, Faculty of Medicino, Kyoto University, Kyoto, Japan, S operated by the Union Carbide Corporation for the United Statos Atomic Energy Commission. ---- . pantom:1*: 1117"eni . 1 " . - Running head: Experiment sa Send Proof to: M. A Bender Biology Division Oak Ridge National Laboratory P. 0. Box Y Oak Ridge, Tennessee 37831 . . ove A -- - . - -- ! SUMMARY The S-4 experiment, "Synergistic Effect of Zero-G and Radiation on Waite Blood Cells", was porformed during the GT-3 manned space flight under an interagency agreement between the National Aeronautics and Space Administration and the U.S. Atomic Energy Commission. Design, fabrication, and testing of the experimental hardware was done by the Oak Ridge 1-12 Plant, while the isotope preparation and biological work was done by the Oak Ridge National Laboratory. . . - - - - • . .-... . .. - The experiment consisted of the simultaneous irradiation of a series of samples of whole, human blood with 32p ß rays during the orbital phase of the mission. Irradiation was accomplished by duplicate experimental devices, one located on the righthand hatch of the space craft and the other on the ground at the launch sito. After completion of the mission a cytogenetic analysis was made of each blood sample, and the frequencies of chromosome aberrations were determined. The yields of both single- and multiple-break aberrations. were calculated for the flight and the ground control portions of the experiment. Comparison of the results showed that while there was no significant difference between the yields of multiple-break aberrations, the frequency of single-break aberrations was significantly higher in the flight samples. Several lines of evidence rule out the possibility that this difference arose from differences in absorbed dose, temperature, oxygen tension, or other parameters known to influence chromosome aberration yields. That the spaceflight by itself induced aberrations is ruled out by the experiment's control blood samples and also by pre. and post-flight blood samples obtained from the right crow. A synergism wuntirinL7! between radiation and some spacefl): 1, parameter thus appears to exist for human chromosome aberration production. It appears likely that this effect is on the normal restitution of chromosome breaks, rather than on chromosome breakage itself. The small positive effect demonstrated by the S-4 experiment is of definito scientific interest. The experiment however failed to confirm the existence of any large offret, such as has previously been suggested by other authors, which might constitute a special obstacle to prolonged manned space flights. INTRODUCTION damage, have been observed following both ballistic and crbital space flights (see, for example, refs. 1 and 2). These effects include mutation, chromosoma aberration production, and cell killing, and in some cases were of many times greater magnitude than would have been predicted from the radiation exposure received during the flight. Such phenomena, if real, would be of obvious concern to manned space flight programs, and also of general interest to radiobiologists. An unpredicted radiobiological effect could be due to either or both of two things. Since one component of the radiation encountered above the earth's atmosphere (the heavy primaries) is not available for test use in terrestrial laboratories, the possibility that these particles have unexpected biological effects must be admitted. It is also possible that other parameters associated with space flight, such as prolonged "weightlessness", interact synergistically with radiation to produce unexpectedly large effects. The 5-4 experiment was prepared in order to test these possibilities, and to settle the question of whether there are, in fact, large radiobiological effects following space flights. The experiment was successfully executed during the GT-3 manned spaceflight on March 23, 1965. EXPERIMENTAL DESIGN A practical plan to test for synergism between radiation and space flight parameters such as "weightlessness" is to irradiate a thoroughly studied biological material with a known quantity and quality of radiation during the "zero-g" phase of an orbital spaceflight, and to compare the types and rates of effects found with those observed in sui.table "in-flight" and "ground" controls (see raf. 3). An increased effect . in the experimental material, including the in-flight control would be evidence of an effect of some space flight, parameter. An increased effect in the experimental material, but not including the in-flight control, on the other hand, would be evidence of a synergism betwoen the radiation administered and some space flight parameter. The substitution of a series of doses and samples in this simple experimental plan allows the determination of dose-effect curves, and the study of any offect observed in torms of the kinetics of the response studied, and, possibly, to elucidation of its mechanism. Such a plan was adopted for the Sky experiment. The production of chromosomal aberrations is one of the best known biological effects of radiation (see ref. 4 for a recent review), and was selected as a suitable response for study. Human leukocytes were selected as the experimental material because of several technical advantages, including the fact that the cells used are all in a uniformly sensitive stage in the cell cycle (G) when whole blood is irradiated, either in vivo or in vitro, and also because our group has had extensive experience with the system (refs. 5 - 10). The small volume and mass which could be alloted to the experimental device aboard the space craft i 2. - ... - - . . precluded the use of X or 8 rays to irradiate the blood samples, primarily because of the shielding which would have been necessary to protect the flight crew. ß rays, which are easily shielded against, were used instead. The radioisotope phosphorous-32 was selected as the source because it emits only a single ß particle, because the particle energy (average q = 0.7 MeV) is suitable, and because she had alroady boon used extensivoly in radiobiology. The use of a liquid phase biological tast material, together with isotopic radiation sources, allowed a "plane parallel" irradiation geometry to be adopted, providing a compact physical arangement giving reasonably homogeneous radiation exposures over the sample volume. Because the induction of chromosome aberrations is a non-linear function of dose, a graded series of four different radiation exposures, plus an unirradiated control, was used. In order to ensure against loss of one or more samples, two complete sets of blood samples were used, with blood from a different donor for each set. The experimental material thus consisted of ton 3-r] whole blond samples, a pair irradiated with each of the radiation doses, and a pair unirradiated as the in-flight control. The simultaneous ground experiment (ground control) consisted of a complete duplicate set of samples drawn from the same donors at the same time as the right material. As an additional in-flight control, -... . - . . . - - - -- -- --- ..:- :-...---.- .... . blood samples were also obtained from the flight crew boforo and after - the mission. ... . .-... .. . .- .... ... - ne . -.- . - EXPERIMENTAL DEVICE The device which was developed to carry out the experimental design is shown in fig.; 1. It consists of a sealed aluminum box, about 9 by 9 by 3.2 cm, with an operating handle protruding from one end. The major components of the device are shown in fig. 2. The exploded, cut-away drawing (fig. 3) shows the arrangement of the parts within the assembled device, with the blood sample holders and opera cing handle shown in the "nonirradiate" position. Each of the five opuxy resin and fiberglass holders contains two sterilo, heparinized, 3-ml blood samples in the form of discs 3 mna thick. The four blood sample holders which are to be irradiated are located in tracks in the aluminum houging running between the paired aluminum and platinum Sp source plate holders. When the operating handle is pushed in these four blood sample holders are moved into position between the pairs of Sep source plates, thus starting the irradiation of the blood samples through the thin blood chamber "windows". When the operating handle is pulled out the blood sample holders are withdrawn, stopping the irradiation. The unirradiated control blood sample holder is located behind, and shielded from the Bap source plate array. The space above the control holder is occupied by an instrument package assembly. The sup source plate pairs are arranged in a graded serios of total activities in a ratio of 1:2:3:4 in order to yield the required series of irradiation doses during the simultaneous exposure of the blood samples. The cross-section drawing (fig.4) shows the geometrical (- rolationships betwoon the 32p sources and the blood samples more clearly.' .... . ::- - -; -- -- - - *-* . . : - .- -.- . • . - - ..- . - 5 A number of measuring devices were included in the experimental device in order to help confirm the correspondence between the in-flight and the ground portions of the experiment. Two silver metaphosphate fluoroglass dosimeter rods were located within the stems of the bicod sample chamber sealing screws (figs. 3 and 4) to record the dose received by each blood sample. An instrument package was designed to provide records of internal device temperature during the experiment, records of excessively high or low temparatures, and a record of the time of irradiation. These records were written by means of £pots of colored iight which moved slowly across strips of color film. Time is read across the resulting stripes on the developed film, while the color at a given point indicates the temperature at that time. The times at which the irradiation is begun and ended are recorded by a separate colorcd spot. The instrument package also contains a pair of large volume silver metaphosphate fluoroglass dosimeter blocks to measure the ambient radiation within the experimental device. A more complete description of the experimental design and of the experimental device and its qualification for flight is given elsewhore (ref. 11). 20 EXECUTION OF THE EXPERIMENT The short cimo over which the blood samples would remain viable, the relatively short half-life of the radioisotope 32p, and the tissue culture procedure required in order to produce chromosome preparations for analysis made the actual execution of the experiment a fairly complex operation. The blcod samples had to be obtained as late as possible. The experimental devices ther: had to be assembisa and tested, and the flight device inserted into the spacecraft shortly before launch. Immediataly after recovery of the spacecraft the flight device had to bo recovered from the spacecraft and opened, and tissue cultures made from each blood sample, as well as from post-flight samples from the flight crew. In order to accomplish these things special facilities had to be provided at the launch site and on board the prime recovery vessel (and on vessels in the first and second orbit recovery areas as well). Preflight Two days before launch sterile peripheral blood samples were obtained from the flight crow (and also from the barkup crow). Short-term loukocyta cultures (ref. 12) were prepared and incubated at 37° C. At . approxdmately nine hours before launch sterile peripheral blood samples were obtained from two pre-tested donors. Two complete experimental -. - devices were assembled and tested. Approximately 210 min before launch . ... the flight device was mounted in an insulated braokat located on the inside . of the right hand hatch of the spacecraft. The ground control dovico was . placed in a controlled tomperature cabinet, the temperature of which was . :.. . . . ------ periodically adjusted to correspond to the temperatures read out from the spacecraft cabin. Fight After the boost phase of the flight the flight device remained essentially weightless until irradiation was started. The irradiation wa's initiated by the pilot 50 min 18 sec after liftoff. Twenty minutes later, at 70 min 18 sec after liftoff the pilot terminated the irradiation. The corresponding activation and deactivation of the ground device occurred at 52 min and 72 min after liftoff, respectively. Except for the minor accelerations of the texas burn" at 1:32:59 G.E.T., the "lateral burn" at 2:16:59 G.E.T., and the "pre-retro burn" at 4:21:23 G.E.T., the in-flight experimental device was essentially weightless for approximately 4 min before irradiation, during irradiation, and for approximately three and one-half hours following irradiation. Postflight Post-flight peripheral blood samples were obtained from the flight crew approximately six and one-half hours after launch. All blood samples were put in culture by ten hours after launch, those from the ground control experimental device at the launch site, and those from the in-flight experimental device, plus the crew post-flight sampies, on the prime recovery vessel. The crew pro-flight cultures were fixed the day after launch, approximately 72 hours after they were made. The experimental cultures, 12. plus the crew post-flight cultures, were all fixed at approximately 77 hours after launch. :-- -: doo.. : RESULTS Post-flight analysis of the fluoroglass dosimeters and other instruments showed that the doses, times of actuation, and temperaturos of the in-flight and ground control experiments agreed well. The instrument package film records agree well with the actuation and temperature information provided by other sources. They indicate that the flight device reached a temperature of 30° C during assembly (during Heliarc welding of the top), and that it did not exceed 380 C nor drop below 140 C at any time during the preflight, flight, or recovery periods. The ground control experimental device never became as warm, remaining between 290 C and 20 c during these periods. Post-flight determinations of circuit element function, battery voltages, and coulome ter gap positions indicate that both instrument packages functioned perfectly. None of the indicators of excessively high or low temperature had been activated. On both the in-flight and the ground control instrument package film records the activation markers appear in the proper position and in that position only, agreeing well with the communications records. That the exposures were of the requires duration is confirmed by the fluoroglass dosimeters incorporated into the blood sample chamber screws. The 4t dosimeters were read on a fluorometer calibrated against fluoroglass standards which had been given known 6°co 8 ray exposures at the National Bureau of Standards. The readings for each pair of dosimeters agreed well. Doses were also estimated pre- and post-flight by means of both fluoroglass and Fricke solution dosimetry. The results 14 are presented in Table 1. The reading values are averaged for the dosimeters from each blood sample. Both the readings and the dose estimates agree well with both the theoretical expectations and the results of previous control experiments. A large part of the approximately two rad dose to the control blood samples was due to Bremsstrahlung .- - ..-. with a peak energy of about 65 KeV. The large volume fluoroglass dosimeter blooks located in the ground control instrument package registered this level of exposure also. Those from the in-flight ... . . instrument package registered somewhat higher exposures. All of the blood sample cultures were successful and yielded satisfactory chromosome preparations. Very little haemolysis was noted after the blood samples had been centrifuged, and no other evidence of gross cell damage was seen. A total of 4,600 cells were analysod for the entire experiment. The results are shown in Table 2. The flight crew chromosome aberration analyses show no increase . - in aberration frequency due to the spaceflight. The two dicentric . chromosomes seen in the samples from one crew member appear to be :. identical, and were without acontric fragments. They therefore cannot be attributed to the spaceflight. ...... The deletion frequencies seen .. - are typioal of normal individuals. Thę aberration frequencies seen in the control blood samples are what is expected in cells exposed to such a low radiation dose (from ſ ray leakage and Bremsstrahlung within the experimental devices), d agree well with what was seen in previous control experiments. The irradiated samples yielded the expected types of aberrations. Figs. 5,. - lii 15 6, and 7 illustrate the two major classes of aberrations induced. The cell shown in fig. 5 is normal, showing 46 chromosomes and no breaks, fragments, or rearrangements. The cell shown in fig. 6 contains a chromosome delotion, with the resulting acentric fragments. Because only one broak 18 required, the yield of this type of aberration 18 a linear function of dose, 'following the expression I = a + bD, fo where I is the yield, D the dose, a the spontaneous frequency, and b the coefficient of aberration production. The cell illustrated in fig. 7 ( contains a dicentric chromosome and the resulting acentric fragments. This class of aberrations, including rings as well as dicentric chromosomes, requires two indepondent chromosome breaks, and the yield is consequently a function of the square of the dose, approximating the expression, 0 - . . .- I = cda fare .. where is the coefficient of dicentricproduction. The data of Table 2 were fitted to these expressions by iterative least-squares regression analyses. The resulting coefficients of aberration production are shown in Table 3, together with those for a typical control experiment made prior to the flight ("Run 5"). The yields for ring and dicentric chromosomes in the in-flight and ground control experiments and for deletions in the ground control experiment agree well with the results of the provious control experiments. The ring 16 and dicentric yields for the in-flight and ground control experiments do not differ significantly from each other. The yield of deletions in the in-flight experiment, however, is roughly twice that seen in the ground control experiment and in the previous control experiments. The difference is significant, and as can be soon from Table 2, completely consistent. : . --...--... :. DISCUSSION The lack of aberrations in the blood samples obtained from the astronauta after the flight nakes the possibility that the radiation encountered during such a spaceflight has an unprecidented effect unlikely, at least for genetic . systems, such as the one used for this experiment. Such a conclusion is strengthened by the low aberration levels seen in the flight control blood samples. All physical evidence contradicts the possibility that the flight and ground control samp) os received significantly different radiation doses. In addition, the aberration data itself shows that the doses were substantially the same. If the high deletion yields from the flight samples were due to their having received a higher dose than the ground control samples, then the ring and dicentric chromosome yields would also have been increased in the flight samples. The possibility that something associated with the spaceflight produced chromosoine breaks independently of the radiation can, of course, be dismissed on several grounds. It appears, then that some spaceflight parameter does in fact interact synergistically with radiation. Since only single break aberrations (deletions) show this effect, it seems likely that the effect is on the chromosome rojoi.ning system rather than on breakage itself. If it were on breakage, the effect would be the same as increasing the dose, and should then affect all aberration classes. Stated differently, the effect would seem to be on the cells' ability to repair damage caused by radiation, rather than on the or amount of damage incurred. Although the effect is not a large one, it is cortainly of great interest from the point of view of radiation cytogenetics. 18 . Further experiments will be necessary, however, in order to confirm the synorgistio effect and to determine just which spaceflight parameter or parametors are involved, and the mechanism of action. ., . ..na's .. :.... ACKNOWLEDGEMENTS The successful execution of the S-4 experiment required the cooperation of a much larger group of people than can possibly be acknowledged individually. We are particularly indebted, however, to H. F. Smith, Jr., W. T. Smith, Jr., and E. N. Rogers of the Oak Ridge Y-12 Plant, to F. N. Case of the ORNL Isotopes Division, to F. M. Faulcon, W. L. Lee, F. J. Pearson, J.Liltongue, K. P. Jones, and J. R. Azzi of the ORNL Biology Division, and to R. M. Rapp of the NASA Manned Spacecraft Center. We also wish to thank Cdr. J. W. Young for his interest and cooperation in the performance of the in-flight portion of the experiment. . . ..... -- .- ... REFERENCES 1. Problemy kosmicheskoy biologii, Vol. I, ed. N. M. Sisakyan, V.S.S.R. Academy of Sciences Publishing House, Moscow, 1962 (NASA TT F-174). 2. Problemy kosmicheskoy biologii, Vol. II, ed. N. M. Sisakyan and vi I. Yazdovskiy, U.S.S.R. Academy of Sciences Publishing House, Moscow, 1962 (JPRS: 18,395; OTS: 63-21437). 3. Position Paper on Theoretical Aspects of Radiobiology as Applied to .. the Space Program, Environmental Biology Committee Panel on Radiation Biology, Space Science Board, NAS-NRC, Washington, 1963. 4. Evans, H. J. Chromosome aberrations induced by ionizing radiations, International Reviews of Cytology 13; 221-321, 1962. 5. Bender, M. A, and P. C. Gooch. Types and rates of X-ray-induced chromosome aberrations in human blood irradiated in vitro. Proc. National Academy of Sciences (Wash) 48; 522-532, 1962. 6. Bender, M. A, and P. C. Gooch. Persistent chromosome aberrations in Irradiated humans. Radiation Research 16; the 53, 1962, and Radiation Research 18; 389-396, 1963. . Bender, M. A, P. C. Gooch, and D. M. Prescott. Aberrations induced in human leukocyte chromosomes by 3H-labeled nucleosides. Cytogenetics 1; 65-74, 1962. Bender, M. A, and P. C. Gooch. Chromatid type aberrations induced by X rays in human leukocyte cultures. Cytogenetics 2; 107-116, 1963. Gooch, P. C., B. A Bender, and M. L. Randolph. Chromosome aberrations . . - .. - - - . - . . . . . . .......... . ........... 8. ....... ................ . induced in human somatic cells by neutrons. IAEA Symposium on the :.: -*. Biological Effects of Neutron Irradiations, Brookhaven National .. Laboratory, Oct. 7-21, 1963. .. . . - 10. Bonder, M. A. Chromosome aberrations in irradiated human subjects. Annals of the New York Academy of Sciences 114; 249-251, 1964. 11. Oak Ridge National Laboratory. Synergistic effect of zero-g and radiation on whito blood colls. An experiment for the Gomini III manned space flight. Annual report, period ending June 30, 1964. ORNL - TM - 940, 1964. 12. Moorhead, P. S., P. C. Nowall, W. J. Mellman, D. M. Battips, and . D. A. Hungerford. Chromosome preparations of leukocytes cultured from human peripheral blood. Experimental Cell Research 20; 613-616, 1960. i s ini TABLE 1 - Dose Estimates From Fluorogla88 Dobimeters Incorporated in . --:'. Experiment Sw4 Blood Sample Chambers. - . . . Device Theoretical dose (rad) . Average reading (ua )* Net B ray dose (MA) Estimate of Bray done (red) Estimated total dose (red) . . . . Ground, control . . 28.1 45 . 92 136 181 4.5 18.1 27.7 41.9 *.51.0 13.6 23.2 37.4 46 Flight device 4,2 52 28.0 29.5 13.8 25.3 38.3 34.1 126 46.8 42.5 157 - :- -- - *As the instrument was set up, one HA equals two Cobo y ray rad. in? .. ... TABLE 2 - Results of Experiment S-4 Chromosome Aberration Analyses. Subject Sample Cells scored Estimated dose (rad) Chromatid Chromosome Ring and deletions deletions dicentric chromosomes Crew 2 0 : 0 Pre-fl 100 Post-fi 200 Pre-f1 100 Post-fl 200 i Experiment Gr 400 2 3 3 400 a , 400 400 400 X Na aa au wt i w 400 400 140 400 128 400 400 159 *These two dicentric chromosomes appear identical; both lacked acentric fragments.. - - TABLE 3 - Coefficients of Aberration Production for S Experiment. Deletions Rings and Dicentrics (x 2014 (x 1063 per cell per rad per cell por rad? Ground Control In-flight 4,79 € 0.72 9.11 $ 1.02 5.36 0.86 3.23 + 0.59 3.48 + 0.53 2.66 + 0,60 Run 5 --. -.- .-- .- me .m a . .-.- - -- - . -- . -. FIGURE LEGENDS Fig. 1. Tho 5.4 experimental devico beforo final sealing. The operating handle is in the "non-irradiate" position. Fig. 2. Major components of the S-4 experimental device showing: 1, operating handle and top assembly; 2 and 3, aluminum housing before welding; 4, set of five fiberglass-reinforced blood sample holders; 5, set of five aluminum and platinump ray source plates; 6, instrument package with shorting plug. F1g. 3. Exploded, cut-away drawing of the experimental device showing the relationships between the component parts. The blood sample holders and the operating handle ero shown in the "non-irradiate" position. Fig. 4. Cross-section drawing of the experimental device showing the ' geometrical relationships between the blood samples and the Bap Bray source plates. The blood sample holders are shown in the "non-irradiato" position. During irradiation the blood sample holders are moved into the vacant spaces betwoon the paired source plates. Fig. 5. Chromosome preparation of a normal unirradiated cell without chromosome breaks, fragments, or rearrangements. Fig. 6. 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