Pergamon

www.elsevier.com/Iocate/asr

Adv. Space Res. Vol. 28, No. 4, pp. 563-568, 2001 cC 2001 COSPAR. Published by Elsevier Science Ltd. All rights reserved

Printed in Great Britain 0273-1177/01 $20.00 + 0.00

Pll: S0273-1177(01 )00392-1

ALKYLATING AGENT (MNU)-INDUCED MUTATION IN SPACE ENVIRONMENT

T. Ohnishi 1", A. Takahashi ~ , K. Ohnishi ~ , S. Takahashi 2, M. Masukawa 2, K. Sekikawa 2, T. Amano 2, T. Nakano 2, and S. Nagaoka 3

~ Department of Biology, Nara Medical University, Kashihara, Nara 634-8521, 2Space Experiment Department, National Space Development Agency of Japan, Tsukuba, lbaraki 305-0047,

~Department of Gravitational Physiology, Fufita Health University School of Health Sciences, Toyoake, Aichi, 470-1192, Japan

# tohnishi @ naramed-u, ac.jp / Fax." + 81- 744-25-3345

ABSTRACT

In recent years, some contradictory data about the effects of microgravity on radiation-induced biological responses in space experiments have been reported. We prepared a damaged template DNA produced with an alkylating agent (N-methyl-N-nitroso urea; MNU) to measure incorrect base-incorporation during DNA replication in microgravity. We examined whether mutation frequency is affected by microgravity during DNA replication for a DNA template damaged by an alkylating agent. Using an b7 vitro enzymatic reaction system, DNA synthesis by Taq polymerase or polymerase III was done during a US space shuttle mission (Discovery, STS-91). After the flight, DNA replication and mutation frequencies were measured. We found that there was almost no effect of microgravity on DNA replication and mutation frequency. It is suggested that microgravity might not affect at the stage of substrate incorporation in induced-mutation frequency. © 2001 COSPAR. Published by Elsevier Science Ltd. All rights reserved.

INTRODUCTION

The effects of space radiation on mutation have been studied in some space experiments. A high frequency of induced mutations was detected in Drosophila (D.) melanogaster after space flight, as compared with the ground control (Ikenaga et al., 1997). Space crews showed chromosomal aberrations in their lymphocytes after space flight (Testard et al., 1996; Obe et al., 1997; Wu et al., 1998). Furthermore, we found that certain space samples gave higher mutation frequencies than those of the ground samples in bacteria and yeast (Yatagai et al., 2000; Fukuda et al., 2000). We assume that DNA damage may be caused by space radiation, because our findings showed an accumulation of p53 in skin and muscle of rats after space flight (Ohnishi et al., 1996, 1999). This suggests that space radiation might induce some types of DNA damage. However, it is still unknown exactly what types of DNA damage are caused /n vivo by space radiation, because microgravity may enhance mutation frequency. The relationship between microgravity and space radiation is controversial, because different results have been reported, i.e., (i) microgravity enhanced radiation effects on abnormal development in Carausius morosus (Bucker et al., 1986) and increased the mutation frequencies in D. melanogaster (Ikenaga et al., 1997), (ii) microgravity decreased radiosensitivity, resulting in promotion of recovery from radiation damage in Deinococcus radiodurans (Kobayashi et al., 1996), (iii) microgravity had no effect on the induced-mutation frequencies in Escherichia coli (Harada et al., 1997) and Dico, ostelium discoideum (Ohnishi et al., 1997; Takahashi et al., 1997) or on repair activity in Saccharomyces cerevisiae (Pross and Kiefer, 1999), E. coli and human fibroblasts (Horneck et al., 1995, 1996, 1997). These contradictory findings may be due to the diversity of experimental systems in the biological experiments flown on spacecrafts. At present, it is very difficult to clearly relate microgravity effect to just one varying factor. Experiments on spontaneous mutation in fruit flies reported that the mutation frequency in space is higher than that on earth (Ikenaga et

563

564 T. Ohnishi et al.

al., 1997). It has been assumed that the high mutation frequency may be induced from incorrect base- incorporation during DNA replication under microgravity environment. However, there is no direct evidence for this assumption. We examined here whether the mutation frequencies are influenced by microgravity using in vitro experiments.

MATERIALS AND METHODS

Experimental Conditions. Plasmid DNA containing an alkylated rpsL region induced by 10 rtg/ml MNU (Nacalai tesque, Kyoto, Japan) for ! h at 37~C was heat-denatured to single-strands and then bound with primers (5 '-CTTCTATrAAGCTTr-CGGACGqTTFACA-3') for DNA synthesis. Two kinds of liquid of 200 ~tl reaction buffer containing these single-stranded plasmid DNA (5 rtg) and 10 ~tl enzyme solution containing Taq polymerase (TAP-211, TOYOBO Co. Ltd., Osaka, Japan)or polymerase III (POL- 301, TOYOBO Co. Ltd.) were separately sealed in compartment a and b, respectively, in a special reaction bag (2.2 cm x 6.0 cm) (Figure 1A).

A

e f g b c, d

B ® ® '+

Fig. 1. Enzymatic reaction system, a, single strand DNA bound with primer and reaction buffer solution; b, blue bead and polymerase; c, empty compartment; d, red bead and reaction stop solution containing detergent, e, f and g, special temporary sealing band. e, marking with red arrow; f, marking with blue arrow. A, before activation; B, after activation; C, after deactivation.

The reaction bag was specially made of polypropylene sheet (Hybrid MekkinBag HM-1304, HOGY, Osaka, Japan) in our laboratory. Space shuttle Discovery (STS-91) was orbiting the earth at an altitude of 400 km and an inclination of 51.6 degrees to earth's equator. The space shuttle Discovery (STS-9 l) was launched from the NASA Kennedy Space Center (Florida, USA) on June 2, 1998. To begin activation, the separating films were broken by pushing from one side of the separated compartment in each bag (Figure 1B). Then, the chemical reaction of DNA synthesis started with mixing of the two kinds of liquid reaction buffer containing single strand DNA and polymerase (Figure 1B). The reaction was incubated at room temperature (21.6-22. I°C) for 26 h. During this period, the synthesis of complementary strands of DNA advanced under microgravity conditions. After incubation of the reaction, further reaction was stopped by breaking the film between compartment c and d and mixing 300 ~tl of stop solution [300 mM NaCI, 20 mM Tris-HC1 (pH 8.0), 20 mM EDTA, 0.6 % sodium dodecyl sulfate (SDS) and 0.2 ~tg/ml proteinase K] (Figure 1C). Two mission specialists (Franklin R. Chang-Diaz, Ph.D. and JanetLynn Kavadi, Ph. D.) on board the space shuttle performed each step of activation and deactivation of the chemical reaction. Finally, the reaction bags were stored in a freezer (-20~). Using blue and red glass beads (,3 mm, No. 5 and 942, TOHO, Osaka, Japan) in each reaction bag; it could be easily confirmed that the two kinds of colorless liquid were mixed. The space shuttle was landed at the Kennedy Space Center on June 12, 1998. Control experiments on earth were performed at the NASA Johnson Space Center (Houston, USA) under almost the same condition after receiving downlink information on the space experimental conditions from the space shuttle. Measurement of DNA Replication Reaction. The template DNA and synthesized DNA which was elongated with 5'-biotin-labeled primer (5 '-CTTCTA-TTAAGCTITCGGACGTITTACA-3') in space,

MNU-lnduced Mutation in Space 565

were purified immediately after landing at SPACEHAB's Payload processing Facility (Florida, USA) with a single extraction of the supernatant using phenol and chloroform. After ethanol precipitation, all samples were loaded as equal amounts of DNA (0.5 ~tg) into the wells of gels in alkali-gel loading buffer (300 mM NaOH, 6mM EDTA, 50% glycerol, 0.15% bromophenol blue). The DNAs were electrophoresed on 1% alkali-agarose gels for 6 h at 30V using alkali-buffer (100 mM NaOH and 2 mM EDTA). Subsequently, the gels were rinsed with neutral buffer (0.5 M Tris-HCl, pH 7.5 and 1.5 M NaC1) and transferred onto GeneScreen Plus membranes (DuPontl NEN Research Products, Boston, MA, USA). The membranes were dried for about 2 h at 80°C. The newly synthesized DNA with 5'-biotin-labeled primer were visualized using horseradish peroxidase-conjugated streptavidin (Calbiochem, La Jolla, CA, USA) and 30 ml of coloring solution ( 15 mg of 4-chloro- 1-naphtol, 5 ml of methanol, 2.5 ml of 0.5 M Tris-HC1, pH 7.5, 22.5 ml of water and 10 ~tl of 30 % H202). Measurement of Mutation Frequency. The ethanol-precipitated DNA was purified by DNA purification Kit (GFX N PCR, Amersham Pharmacia Biotech. Inc., NJ, USA). The DNA was cut with a restriction enzyme (BspLU 1 l I, Boehringer Mannheim, Germany). All samples were loaded in each gel slot with gel-loading buffer (final concentration at 0.004 % bromophenol blue, 0.2 % SDS and 10 % glycerol). The DNA bands containing rpsL region were separated by 3 % agarose gel electrophoresis (40 mM Tris-acetate, 2 mM EDTA and 0.5 lag/ml ethidium bromide) using Mupid-2 (COSMO Bio Co., Ltd., Tokyo, Japan) and purified by DNA purification Kit. The blunted DNA by Blunting high (TOYOBO Co. Ltd.) and Eco RI adopter (TAKARA, Shiga, Japan) were ligated by Ligation high (TOYOBO Co. Ltd.). These insert DNA and Eco RI treated ~,ZAPII phage DNA (Stratagene, CA, USA) were ligated by Ligation high. The ligated DNA samples were packaged by Gigapack III Gold (Stratagene) and constructed for in vivo excision of pBluescript SK (-) phagemid vector by )~ZAPII Vector Kits (Stratagene). The selected DNA fragment newly synthesized in space by cutting with a restriction enzyme (Act/I, TOYOBO Co. Ltd.) were separated by 3 % agarose gel electrophoresis and purified by DNA purification Kit. The selected DNA was electroporated using a CELL-PORATOR (GIBCO BRL, Life Technologies. Inc., Rockville, MD, USA) into E. coli (DH1 IS) cells as an indicator. To select transformants, E. coli cells were spread both on NZY (0.5% NaC1, 0.2% MgSO47H20, 0.5% yeast extract, 1% casein hydrolysate, pH7.5) plates containing 100 gg/ml 5-bromo-4-chloro-3-indolyl-/3-D-galactoside (X-gal, Nacalal tesque), 0.5mM isopropyl-/3-D-thiogalactopyranoside (IPTG, Nacalai tesque) and 100 ~tg/ml ampicillin (Ap, Meiji Seika Kaisha, Ltd., Tokyo, Japan) and on NZY plates containing 100 gg/ml X-gal, 0.SmM IPTG, 100 gg/ml Ap plus 100 ~tg/ml streptomycin (Sm, Meiji Seika Kaisha, Ltd.) after electroporation with pBluescript SK (-) phagemid vectors. After incubation at 37°C for 20 h, bacteria carrying pBluescript SK (-) without an insert form blue colonies, while bacteria carrying recombinant clones with an insert form white colonies (Nakano et al., 1995). Therefore, we can select transformants carrying recombinant clones with rpsL by blue/white selection methods. The mutation frequency of rpsL gene is the ratio of Ap and Sm resistant colonies to Ap resistant colonies. Statistical Analysis. Levels of significance were calculated using the unpaired Student's t-test. P<0.05 was taken to indicate a significant difference.

R E S U L T S

1 2 M 3 4 M 5 6 M 7 8

bp

2238

580 P.

342

b

Fig. 2. The length of DNA chain synthesized in space and on earth. Lanes 1-4, Taq polymerase; lanes 5-8, polymerase III; lanes 1,2,7 and 8, on earth; lanes 3-6, in space; lane M, molecular marker.

566 T. Ohnishi et al.

DNA Replication Reaction. The DNA synthesis during space flight was analyzed by Southern blotting (Figure 2). Newly synthesized products by incubation of Taq polymerase or polymerase III at room temperature (21.6-22. I~C) for 26 h were ca 0.6 and 1.0 kbp, respectively. No clear difference in the length and content of newly synthesized products between space and the control ground samples was found Figure 2). Although the content of newly synthesized products against alkylated DNA was a few than that against untreated DNA, there was no difference in length of the product against both DNAs (data not shown).

Mutation Frequency. The effect of microgravity on induced-mutation through DNA synthesis during space flight was studied (Figure 3). The mutation frequency of alkylated DNA and untreated DNA as control through DNA synthesis by Taq polymerase were 3.1 ± 1.0 and 0.9 ± 0.2 on earth and 3.0 ± 0.7 and 0.8 _+ 0.1 in space, respectively (Figure 3A). Similarly, the mutation frequency of alkylated DNA and the control DNAby polymerase III were 2.4 ± 1.2 and 0.5 ± 0.2 on earth and 2.4 ± 1.1 and 0.6 ± 0.3 in space, respectively (Figure 3B). There was no significant difference in the mutation frequency between space and the control ground samples (P>0.05).

o 4 X

o 3 t -

2 l - -

i t

t -

.o 1

0

f B

Fig. 3. The induced-mutation of newly synthesized DNA. Closed column, on earth; open column, in space.

10 0 10 MNU (l~g/rnl)

A, Taq polymerase; B, polymerase IlL

D I S C U S S I O N

In S/MM-9 flight, we performed to determine the effect of microgravity on the induced-mutation using an in vitro enzymatic reaction system (Figure 1). In space, it is difficult to take exact volume of chemical solution and to mix chemical solutions in handling. Based on the valuable information gained thereby, our experiment was completely successful, and we obtained the enough samples to examine the effects of microgravity on in vitro mutation frequency of damaged DNA. The present space experiments were dependent on new experimental instruments to measure exact volumes of chemical solutions. When we aim for successful space experiments, it is important to have excellent instruments, because space is a microgravity environment. Thus, we cannot use regular instruments. In addition, the specially devised bags were low cost, compact, lightweight and convenient even if a large number of samples must be processed during space flight. We monitored the DNA replication reaction using Southern blotting analysis (Figure 2). The results indicate that there is almost no effect of microgravity on the DNA replication in space (Figure 2). This suggests that the space environment may not influence DNA replication. We obtained the rpsL region in order to measure the induced-mutation frequency of the replicated DNA in space. We measured the number of mutants of in vitro system (Figure 3). The results clearly showed that there was almost no difference in mutation frequencies between the space and the control ground samples, when we used damaged or undamaged DNA as a template (Figure 3). It is suggested that microgravity might not influence an any stage of DNA synthesis through substrate incorporation in induced-mutation frequency. We also found no significant effects of microgravity on the

MNU-lnduced Mutation in Space 567

ligation of damaged DNA during space flight using in vitro systems (Takahashi et al., 2000). These findings support the results obtained from previous space experiments (Harada et al., 1997; Ohnishi et al., 1997; Takahashi et al., 1997; Pross and Kiefer, 1999; Horneck et al., 1995, 1996, 1997). In future studies, therefore, the effects of microgravity on mutation induced by space radiation should be studied. The studies may provide useful data in determining how to protect crew health during prolonged stays in space from carcinogenesis. Furthermore, to clarify the effects of microgravity on mutation frequency, researchers should perform experiments under microgravity and lg in space laboratories. In addition, more complex systems such as higher organisms are necessary in future, because we applied here simple in vitro experiments.

ACKNOWLEDGMENTS

The successful completion of space experiments is only possible with the contribution of many peoples. We thank all the members of our laboratory for their enthusiastic help. The staff of NASDA, JSUP, TRC, NASA and SPACEHAB provided not only perfect organization and optimal support, but also a pleasant working atmosphere. The data were obtained from the joint NASA and NASDA RRMD program.

REFERENCES

Bucker, H., G. Horneck, G. Reitz, E. H. Graul, H. Berger, H. Hoffken, W. Ruther, W. Heinrich, and R. Beaujean, Embryogenesis and organogenesis of Carausius morosus under spaceflight conditions. Naturwissenschaften, 7 3, 433-434, 1986.

Fukuda, T., K. Fukuda, A. Takahashi, T. Ohnishi, T. Nakano, M. Sato, and N. Gunge, Analysis of deletion mutation of the rpsL gene in the yeast Saccharomyces cerevisiae detected after long-term flight on the Russian space station MIR. Mutat. Res., 470, 125-132, 2000.

Harada, K., Y. Obiya, T. Nakano, M. Kawashima, T. Miki, Y. Kobayashi, H. Watanabe, K. Okaichi, T. Ohnishi, C. Mukai, and S. Nagaoka, Cancer risk in space due to radiation assessed by determining cell lethality and mutation frequencies of prokaryotes and a plasmid during the Second International Microgravity Laboratory (IML-2) Space Shuttle experiment. Oncol. Report, 4, 691-695, 1997.

Horneck, G., P. Rettberg, M. Schafer, H. Zimmermann, H. Rink, C. Baumstark-Khan, and S. Kozubek, Enzymatic repair of radiation-induced DNA damage under microgravity, in Radiation Research 1895- 1995, Vol.2, eds. U. Hagen, D. Harder, H. Jung, and C. Streffer, pp. 1195-1198, Universitatsdruckerei, Wurzburg, 1995.

Horneck, G., P. Rettberg, C. Baumstark-Khan, H. Rink, S. Kozubek, M. Schafer, and C. Schmitz, DNA repair in microgravity: studies on bacteria and mammalian cells in the experiments REPAIR and KINETICS. J. Biotechnol., 4 7, 99-112, 1996.

Horneck, G., P. Rettberg, S. Kozubek, C. Baumstark-Khan, H. Rink, M. Schafer, and C. Schmitz, The influence of microgravity on repair of radiation-induced DNA damage in bacteria and human fibroblasts. Radiat. Res., 147, 376-84, 1997.

Ikenaga, M., I. Yoshikawa, M. Kojo, T. Ayaki, H. Ryo, K. Ishizaki, T. Kato, H. Yamamoto, and R. Hara, Mutations induced in Drosophila during space flight. Biol. Sci. Space, 1 1,346-350, 1997.

Kobayashi, Y., M. Kikuchi, S. Nagaoka, and H. Watanabe, Recovery of Deinococcus radiodurans from radiation damage was enhanced under microgravity. Biol. Sci. Space, 1 0, 97-101, 1996.

Nakano, T., K. Okaichi, K. Harada, H. Matsumoto, R. Kimura, K. Yamamoto, S. Akasaka, and T. Ohnishi, Mutations of a shuttle vector plasmid, pZ189, in Escherichiacoli induced by boron neutron captured beam (BNCB) containing a-particles. Mutat. Res., 3 3 6, 153-159, 1995.

Obe, G., I. Johannes, C. Johannes, K. Hallman, G. Reitz, and R. Facius, Chromosomal aberrations in blood lymphocytes of astronauts after long-term space flights. Int. J. Radiat. Biol., 72, 727-734, 1997.

Ohnishi, T., N. Inoue, H. Matsumoto, T. Omatsu, Y. Ohira, and S. Nagaoka, Cellular content of p53 protein in rat skin after exposure to a space environment. J. Appl. Physiol., 8 1, 183-185, 1996.

Ohnishi, T., A. Takahashi, K. Okaichi, K. Ohnishi, H. Matsumoto, S. Takahashi, H. Yamanaka, T. Nakano, and S. Nagaoka, Cell growth and morphology of Dict3'ostelium discoideum in space environment. Biol. Sci. Space, 1 1, 29-34, 1997.

Ohnishi, T., A. Takahashi, X. Wang, K. Ohnishi, Y. Ohhira, and S. Nagaoka, Accumulation of a tumor suppressor p53 protein in rat muscle during a space flight. Mutat. Res., 430, 271-274, 1999.

Pross, H. D., and J. Kiefer, Repair of cellular radiation damage in space under microgravity conditions. Radiat. Envir. Biophys., 38, 133-138, 1999.

568 T. Ohnishi et al.

Takahashi, A., K. Ohnishi, M. Fukui, T. Nakano, K. Yamaguchi, S. Nagaoka, and T. Ohnishi, Mutation frequency of Dictyostelium discoideum spores exposed to the space environment. Biol. Sci. Space, 1 1, 81-86, 1997.

Takahashi, A., K. Ohnishi, S. Takahashi, M. Masukawa, K. Sekikawa, T. Amano, T. Nakano, S. Nagaoka, and T. Ohnishi, The effects of microgravity on ligase activity in DNA repair of double- strand breaks. Int. J. Radiat. Biol., 7 6, 783-788, 2000.

Testard, I., M. Ricoul, F. Hoffschir, A. Flury-Herard, B. Dutrillaux, B. Fedorenko, V. Gerasimenko, and L. Sabatier, Radiation-induced chromosome damage in astronauts' lymphocytes, Int. J. Radiat. Biol., 70, 403-411, 1996.

Wu, H., R. K. Sachs, and T. C. Yang, Radiation-induced total-deletion mutations in the human hprt gene: a biophysical model based on random walk interphase chromatin geometry. Int. J. Radiat. Biol., 73, 149-156, 1998.

Yatagai, F., T. Saito, A. Takahashi, A. Fujie, S. Nagaoka, M. Sato, and T. Ohnishi, rpsL mutation induction after space flight on MIR. Mutat. Res., 4 5 3, 1-4, 2000.