Department of Surgery, University of Medicine and Dentistry of New Jersey, School of Osteopathic Medicine, Stratford, New Jersey 08084
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ABSTRACT |
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The objectives of this study were to assess oxidant
damage during and after spaceflight and to compare the results against bed rest with 6° head-down tilt. We measured the urinary excretion of the F2 isoprostane, 8-iso-prostaglandin (PG)
F2, and 8-oxo-7,8-dihydro-2 deoxyguanosine (8-OH DG)
before, during, and after long-duration spaceflight (4-9 mo) on
the Russian space station MIR, short-duration spaceflight on the
shuttle, and 17 days of bed rest. Sample collections on MIR were
obtained between 88 and 186 days in orbit. 8-iso-PGF2
and 8-OH DG are markers for oxidative damage to membrane lipids and
DNA, respectively. Data are mean ± SE. On MIR, isoprostane levels
were decreased inflight (96.9 ± 11.6 vs. 76.7 ± 14.9 ng · kg
1 · day
1,
P < 0.05, n = 6) due to decreased dietary intake
secondary to impaired thermoregulation. Isoprostane excretion was
increased postflight (245.7 ± 55.8 ng · kg
1 · day
1,
P < 0.01). 8-OH DG excretion was unchanged with spaceflight and increased postflight (269 ± 84 vs 442 ± 180 ng · kg
1 · day
1,
P < 0.05). On the shuttle, 8-OH DG excretion was
unchanged in- and postflight, but 8-iso-PGF2
excretion
was decreased inflight (15.6 ± 4.3 vs 8.0 ± 2.7 ng · kg
1 · day
1,
P < 0.05). No changes were found with bed rest, but
8-iso-PGF2
was increased during the recovery phase (48.9 ± 23.0 vs 65.4 ± 28.3 ng · kg
1 · day
1,
P < 0.05). The changes in isoprostane production were
attributed to decreased production of oxygen radicals from the electron
transport chain due to the reduced energy intake inflight. The
postflight increases in the excretion of the products of oxidative
damage were attributed to a combination of an increase in metabolic
activity and the loss of some host antioxidant defenses inflight. We
conclude that 1) oxidative damage was decreased inflight, and
2) oxidative damage was increased postflight.
isoprostanes; 8-hydroxydeoxyguanosine
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INTRODUCTION |
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THERE ARE A NUMBER OF REASONS for suspecting that oxidative stress may be increased with spaceflight. There is an increase in the exposure to high-energy radiation because of the absence of the protective effects of the earth's atmosphere, with the resultant generation of high-energy free radicals (25). Other possible causes for increased free-radical generation are altered oxygen metabolism from perturbed gas exchange within the lungs (38), or a change in intermediary metabolism. The Skylab investigators suggested that a possible reason for an apparent increase in energy expenditure during spaceflight was an uncoupling of oxidative phosphorylation (28). A similar suggestion was made by Burakhova and Mailyan after examining the electron transport system in rat skeletal muscle after 3 wk in space (3). The body generates ~5 g of reactive oxygen species per day, mostly by leakage from the electron transport chain during oxidative phosphorylation (12).
Until recently, the quantification of oxidative stress has been
difficult to assess in humans because of the lack of sensitive and
reliable assays. This problem now appears to have been solved by the
discovery of the F2 isoprostanes to assess free-radical lipid oxidation and the development of methods for the analysis of the
products of DNA oxidation, specifically 8-oxo-7,8-dihydro-2 deoxyguanosine (8-hydroxydeoxyguanosine, 8-OH DG) (20, 24, 29).
Isoprostanes are derived from arachidonic acid containing phospholipids
by autooxidation, leading to a series of PGF2-like compounds. The bicylcoendoperoxide PG intermediates are reduced to four
regioisomers, each of which can comprise eight racemic diastereoisomers. These 64 isomers are collectively called the PGF2
isoprostanes [8-iso-prostaglandin (PG)
F2
].
Like lipids, DNA is also susceptible to oxidative damage (1, 7, 27); in
fact, DNA is constantly being damaged and repaired in living cells. It
has been estimated that the damage rate is ~104
nucleotides · cell1 · day
1
(20). The most abundant of the nucleotide oxidation products is
8-hydroxydeoxyguanosine. Once produced, 8-hydroxydeoxyguanosine is not
further degraded and is excreted in the urine without further metabolism (19). Measurements of urinary isoprostane and
8-hydroxydeoxyguanosine excretion have provided strong supporting
evidence for a role for oxidative damage in the pathogenesis of a wide
variety of human disorders, including atherosclerosis and cancer
(11, 20, 21, 24).
The primary objectives of this experiment were to assess oxidant stress
during and after long-duration spaceflight on the Russian space
station, MIR. To assess oxidative damage, we measured the urinary
excretion of 8-iso-PGF2 and 8-hydroxydeoxyguanosine. Together the two assays can provide an assessment of any oxidative damage incurred. To facilitate interpretation of the data from MIR, we
compared the MIR data against the results from a short-duration (17-day) shuttle mission and a 6° head-down tilt, bed- rest study.
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METHODS |
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Informed consent forms were obtained in accordance with the policies of the United States National Aeronautics and Space Administration (NASA), the Russian Academy of Sciences Experiment Board, The Russian Space Agency (RSA), and the University of Medicine and Dentistry of New Jersey.
Long-Duration Flight on MIR
The subjects for this study were two astronauts (American) and four cosmonauts (Russian). Time in earth orbit varied with the subject; range was 4-9 mo.Pre- and postflight. The preflight measurements consisted of between two and four sessions during the year before the mission. Each session lasted 2 days. Twenty-four-hour pools were obtained on day 2, and for most sessions 24-h pools were also collected on day 1 of the session. The actual number of preflight sessions depended on crew availability. Sessions were conducted either at NASA facilities in the US (the Johnson and Kennedy Space Centers) or the RSA facility at Star City, near Moscow. During each of the two days, the subjects kept a detailed record of their dietary intake.
The American astronauts were launched and landed on the shuttle in Florida; however, before launch they lived in Russia, in RSA facilities in Star City. They were flown to Houston and the postflight studies were continued at the Johnson Space Center. The Russian cosmonauts returned on a Soyuz space vehicle and spent the first 2 wk postflight at Star City. The postflight studies followed the same protocol as the preflight, with sessions being either return (R)+0 and R+1, or R+1 and R+2, R+6 and R+7, and R+13 and R+14. On occasion, a session was displaced by a day due to a crew member's being unavailable. For the preflight period, each session entailed 2 days of dietary monitoring with 24-h urine collections onInflight.
With two exceptions, a similar protocol to that of the
preflight and postflight periods was performed inflight. All of the inflight data were collected between 88 and 147 days of spaceflight [mean 147 ± 8 days (33)]. Briefly, the food was bar
coded, and the crew recorded the time and amount of food eaten.
Opportunities for collecting 24-h urines on MIR were limited because of
the need to conserve water; only one 24-h urine collection could be done at a time, because on MIR, urine water was recycled for future use. Inflight urine collections were done between 88 and 147 days after
launch. The urines were collected in specially designed plastic bags.
An aliquot was removed and put in a 10-ml syringe, which was placed in
the on-board 20°C freezer. Samples were brought back to
earth on the shuttle supply missions and stored at
70°C until analyzed (details given in Ref. 33).
Diet analysis. The dietary records were analyzed by NASA personnel from the Nutrition and Metabolism Laboratory of the Johnson Space Center by use of the Nutritionist 2.8 program (University of Minnesota, St. Paul, MN), together with some data especially collected by NASA personnel on Russian foods.
Space Shuttle
The urine samples used for this study were collected on the 17-day Life and Microgravity mission (LMS), which was flown in the summer of 1996. Details of the mission and the sample collection are given in Ref. 34. Briefly, the subjects were the four payload crew members of the LMS mission. There were two overall goals of the mission. The first was to conduct a series of experiments on the response of the human musculoskeletal system to spaceflight, and the second was to perform a series of material science experiments. The samples analyzed for this experiment were the urines collected as part of the nitrogen and energy balance studies (34). Daily dietary intake was monitored, and daily urine collections were made for the period beginning 15 days before launch and ending 15 days after landing, as previously described (34). Samples were stored atBed Rest
A 17-day bed-rest study with 6° head-down tilt was conducted in the Clinical Research Center of the NASA-Ames Research Center with eight healthy adult males recruited from the local community. The objective of the bed-rest study was to compare bed rest and spaceflight, using the LMS mission as the flight study. Accordingly, the bed-rest study was designed to simulate the flight experiment as closely as possible. The bed-rest study included the full complement of exercise testing that was done on the shuttle payload (34).As with the flight study, the bed-rest study was divided into three
phases, a 15-day pre-bed-rest ambulatory period, followed by 17 days of
bed rest, and ending with a 15-day recovery period. During the 47 days
of the study, the subjects received all their food from the research
center. An attempt was made to provide the subjects with a
"controlled" ad libitum diet. Twelve daily menus were made up,
comprised of 2,500 kcal/day and 90 g protein/day. In addition, subjects
were allowed access to a snack basket containing fruit, cookies, some
candy, and granola bars. Details of the bed-rest study are given in
Refs. 31 and 34. Urine was collected continuously for the 47-day period
and kept frozen at 70°C until analyzed.
Analytical methodology. In some cases where a 24-h pool was missing from the MIR studies, the missing pool was supplied by NASA as part of a sample-sharing agreement between investigators. Most of the urinary creatinine values for the MIR studies were supplied by NASA; the remainder were measured by us using the picric acid method with a kit marketed by Sigma-Aldrich (St. Louis, MO). The 24-h pools for the LMS mission were available from our previous experiments (31, 34). Accurate 24-h pools were not available for the bed-rest study because all urine voids were ad libitum; therefore, some of the potential "24"-h pools deviated quite significantly from 24 h, so either 48- or 72-h pools were made up by the fractional aliquot method. Where the number of periods was uneven (e.g., the bed-rest period was actually 17 days), the paired days were selected so that the 2-day exercise periods fell in the same pool (31).
Isoprostane analyses were done on unextracted urine (UN) and an organic extract of urine (EX), by use of an ELISA kit (Oxford Chemicals, Oxford, MI). The isoprostanes were extracted from the urine using the methodology recommended by the manufacturer. Urine (0.5-1 ml) was adjusted to pH 3.0 and loaded onto a C18 Sep-Pak (Waters, Milford, MA). After the sample was washed with water (10 ml), followed by heptane (10 ml), the isoprostanes were eluted with ethyl acetate (5 ml). Sodium sulfate (~1 g) was added, and the solution was applied to a silica Sep-Pak and the isoprostanes eluted with a 1:1 mixture of ethyl acetate and methanol. The solvent was removed with dry N2, and the residue was dissolved in a known volume of a dilution buffer supplied by the kit manufacturer and assayed by ELISA. For the MIR isoprostane samples, each sample (extracted or unextracted) was assayed at three different dilutions. For 8-OH DG we used the ELISA kit (Genox, Baltimore, MD). Each sample from MIR was assayed in triplicate at three dilutions. For financial reasons the LMS flight and bed-rest studies were analyzed only for 8-iso-PGF2Statistics. Data were analyzed by a repeated-measures design ANOVA (RMANOVA). For consistency all data sets were divided into three time periods, preflight (pre-bed-rest), flight (bed rest), and postflight (recovery), and the natural logarithms of the data were used for the RMANOVA. Significance was accepted at P < 0.05. If the RMANOVA indicated significance at P < 0.05 or better, group differences were identified by the Student-Newman-Keuls test. To investigate whether there was any time dependence in the postflight (bed-rest) period, we used paired t-tests of a specific postflight (bed-rest) period against the mean preflight value. Significance was accepted at P < 0.01. The Sigmastat Statistical System (SPSS, Chicago, IL) was used for the statistical computations. Data in the text, figures, and tables are means ± SE.
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RESULTS |
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Regarding MIR, we have previously described the changes in nutrition
and the accompanying weight changes for these astronauts before and
after spaceflight (33). Briefly, the average weight loss was 4.6 ± 1.1 kg (range 2.4-8.0 kg, Table 1).
Energy intake inflight was significantly less than either pre- or
postflight (22 ± 9%, P < 0.05, Table 1). Postflight energy
intake returned to, but was not increased over, the preflight levels.
Likewise, dietary intake of the anti-oxidant vitamins (A, C, E, and
selenium) were similar pre- and postflight (Table
2). We have no data on antioxidant intake
inflight (Table 2). The urinary excretion of 8-iso-PGF2
was decreased by 20% inflight (P < 0.05); 8-OH DG was
unchanged (Table 1).
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The postflight data collection schedule called for three sessions of 2 days per session, beginning on R+0 or R+1, R+6 or R+7 and R+13 or R+14.
The sessions were combined to give a single data set for each subject.
Isoprostane levels were decreased inflight by ~20% and increased
postflight by 200% (Table 1, P < 0.01). These relationships
applied to both UN and EX and whether the results were expressed as per
kilogram body wt or normalized to creatinine (Table 1). There was no
change in 8-OH DG inflight, but as with 8-iso-PGF2, 8-OH
DG excretion was substantially increased postflight (Table 1, P < 0.05). Figure 1 shows the postflight
time course for 8-OH DG and 8-iso-PGF2
. Excretion of
8-iso-PGF2
was substantially increased for the duration of the postflight measurement period.
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As for the shuttle, the dietary data in Table
3 are for the days corresponding to the
urine samples analyzed, namely five samples from the week immediately
preceding launch, the five last samples from the flight period, and the
first five days after landing. Energy intake was decreased by 40%
inflight. As with MIR, postflight energy intake returned to, but was
not increased over, the preflight levels. No changes in 8-OH DG
inflight or postflight were found; however, isoprostane levels in
unextracted urine were decreased inflight by ~40% (Table 3,
P < 0.01). No changes postflight were observed, although the
error bars are much larger postflight than preflight (Fig.
2).
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Energy intake was reduced by 10% during the bed-rest period (Table
4) (34). Excretion of 8-OH DG was unchanged
during and after bed rest. Excretion of 8-iso-PGF2
(extracted and unextracted) was unchanged during bed rest but was
increased during the recovery phase by ~30% (Table 4). Statistical
significance was found only with the data normalized to creatinine. As
with the other two studies, the SEs are much greater, particularly for
8-iso-PGF2
during the recovery phase (Fig.
3).
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DISCUSSION |
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The database. Although there was considerable negative publicity in the lay press about the problems encountered with the Shuttle-MIR program, these flights (MIR 21 and 23) were less problematic than some of the earlier or later flights (4). As described in our previous paper, although the amount of data obtained was not large, the quality of the data was good (33). The principal reasons for the limited data were that 1) one of the Russian crew members was switched preflight for medical reasons; 2) technical problems on MIR precluded any measurements during the first three months in orbit; and 3) crew availability for all three phases of the experiment was limited (4).
There were also limitations for the shuttle and bed-rest studies. For the shuttle, the amount of urine available was inadequate to permit assays for both extracted and unextracted 8-iso-PGF2Inflight.
On both MIR and the space shuttle, 8-iso-PGF2 was
decreased inflight (Tables 1 and 3). The decrease was found with both
EX and UN for MIR and for UN on the shuttle. With both missions, 8-OH
DG showed a weak trend toward an increase (Figs. 1 and 2). The two
conclusions that can be drawn with certainty from the inflight results
are 1) that the decreased isoprostane excretion on MIR is not
an artifact caused by possible environmental abnormalities on MIR,
because the same result was found on the space shuttle, and 2)
oxidative damage to lipid membranes was not increased during spaceflight. The discussion of the inflight data that follows is less
certain but is reasonable.
Postflight.
Oxidative damage was increased after spaceflight on MIR. Both 8-OH DG
and 8-iso-PGF were increased by more than twofold after 3+ mo on MIR
(Table 1, Fig. 1). 8-iso-PGF2
was also increased by a
lesser amount after bed rest (Table 4), but not after 17 days on the
space shuttle (Table 3).
Radiation. This study found no evidence for increased free-radical production from high-energy radiation. The radiation flux ranged between 30 and 100 mREM/day with a mean of 60 mREM/day. During these flights there were no bursts of high-energy radiation (personal communication, Dr. V. S. Schneider). Free-radical production from ionizing radiation is qualitatively and quantitatively different from that of metabolic origin. A "hit" from a high-energy particle is an infrequent event compared with the continuous flux of low-energy semistable free radicals generated from metabolic processes. But the collateral damage from the impact of a single high-energy incoming particle can be extensive because of the very high energies involved. The weak trend toward an increase in 8-OH DG production inflight can be accounted for by the decreased synthesis of host defense proteins secondary to the overall depression of protein synthesis.
Different results may be found with other missions in different orbits exposed to different degrees of solar and extragalactic radiation and where nutritional status is not a confounding variable. In the present study, any radiation-induced damage could have been obscured by the metabolic decrease in free-radical production. Any apparent benefit from the "protective effects" of undernutrition against oxidative damage is counterbalanced by the much more serious consequences of undernutrition. In summary, 1) oxidative damage was decreased during long-duration spaceflight on MIR secondary to an overall decrease in metabolic activity; 2) inflight, undernutrition may have a protective effect against oxidative damage; and 3) oxidative damage was increased after return from several months in earth orbit. ![]() |
ACKNOWLEDGEMENTS |
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Special thanks are due to the astronauts and cosmonauts who participated in this experiment. We would like to thank Robert Pietrzyk, M.S. (Wyle Laboratories), for acting as experiment manager, Barbara Rice, R.D. (Wyle Laboratories), for the dietary data, and Scott M. Smith, Ph.D. (NASA-JSC) for coordinating the project, as well as numerous unnamed people at NASA and the RSA who did much of the work in collecting the data in Russia and in the US. M. R. Donaldson provided technical assistance with some of the analyses. Finally, we wish to acknowledge a helpful discussion with Dr. Karl Kirsch, Free University of Berlin, on thermoregulation during spaceflight.
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FOOTNOTES |
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This study was supported by National Aeronautics and Space Administration contract no. NAS9-19409, National Institutes of Health Grant #RO1-14098, and internal UMDNJ funds.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: T. P. Stein, Dept. of Surgery, Univ. of Medicine and Dentistry of New Jersey, School of Osteopathic Medicine, 2 Medical Center Drive, Stratford, NJ 08084 (E-mail: tpstein{at}umdnj.edu).
Received 14 April 1999; accepted in final form 12 October 1999.
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