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Adv. Space Res. Vol. 28, No. 4, pp. 555-561, 2001 © 2001 COSPAR. Published by Elsevier Science Ltd. All rights reserved

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PIE S0273-1177(01 )00391-X

THE EFFECTS OF MICROGRAVITY ON INDUCED MUTATION IN ESCHERICHIA COLI AND SACCHAROMYCES CEREVISIAE

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

1Department of Biology, Nara Medical University, Kashihara, Nara 634-8521, 2Space Experiment Department, National Space Development Agency of Japan, Tsukuba, Ibaraki 305-0047,

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

~ tohnishi @ naramed-u.ac.jp ~Fax.'+81-744-25-3345

ABSTRACT

We examined whether microgravity influences the induced-mutation frequencies through in vivo experiments during space flight aboard the space shuttle Discovery (STS-91). We prepared dried samples of repair- deficient strains and parental strains of Escherichia (E.) coli and Saccharomyces (S.) cerevisiae given DNA damage treatment. After culture in space, we measured the induced-mutation frequencies and SOS- responses under microgravity. The experimental findings indicate that almost the same induced-mutation frequencies and SOS-responses of space samples were observed in both strains compared with the ground control samples. It is suggested that microgravity might not influence induced-mutation frequencies and SOS-responses at the stages of DNA replication and/or DNA repair. In addition, we developed a new experimental apparatus for space experiments to culture and freeze stocks of E. coli and S. cerevisiae cells.

© 2001 COSPAR. Published by Elsevier Science Ltd. All rights reserved.

INTRODUCTION

In space, astronauts are continuously exposed both to radiation and microgravity. In recent years, physical monitoring of space radiation has detected about 1 mSv per day at low-Earth-orbit (Doke et al., 1995). In related experiments, it has been reported that the space station Mir is also exposed to low levels of space radiation of about 0.25-0.51 mGy/day at high-Earth-orbit (personal communication from Dr. H. Yasuda, National Institute of Radiological Sciences, Chiba, Japan). Risk models for astronauts who are exposed to space radiation consisting of vastly different types and energies must be developed to assure adequate protection in future space flight missions. Chronic exposure to space radiation at low dose-rates will be the ultimate limiting factor for an astronaut's career exposure (Cucinotta and Wilson, 1995). The issue of whether the biological effects of space radiation are modified by microgravity is important for risk estimation from a point of view of relative biological effectiveness (RBE) of space radiation. However, 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 Drosophila 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 E. coli (Harada et al., 1997) and Dictyostelium discoideum (Ohnishi et al., 1997; Takahashi et al., 1997), or on repair activity in S. cerevisiae (Pross and Kiefer, 1999), E. coli and human fibroblasts (Horneck et al., 1996). These contradictory results 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 these different effects to just one varying factor. Here, we have aimed to determine the effects of microgravity aboard space shuttle Discovery (STS-91) on induced-mutation frequencies and SOS-responses through the depression of repair processes. We used strains of E. coIi and S. cerevisiae defective in excision repair, recombinational repair and inducible repair, and their wild type strains. These organisms have been used as in vivo model systems for the study of fundamental processes in induced-mutation.

555

556 A. Takahashi et al.

M A T E R I A L S A N D M E T H O D S

Bacteria and Yeast Cells. The plasmid DNAs, YEp51-KS containing wild-type rpsL (provided by Dr. Gunge, Kumamoto Institute of Technology, Japan) or pSK1002 containing umuC-'lacZ (provided by Dr. Akasaka, Osaka Prefectural Institute of Public Health, Japan) were introduced into E. coli and S. cerevisiae (Table 1). Three E. coli strains carrying different DNA repair capabilities (wild-type, KY390; UV-sensitive, KY396; alkylating agents-sensitive, KT222) were irradiated with UVC or X-rays or treated with 1-methyl- 1-nitrosourea (MNU). Four S. cerevisiae strains carrying different DNA repair capabilities (wild type, YEWV and HYT223; UV-sensitive, YESV; ionizing radiation-sensitive, HYT239) were irradiated with UVC or X-rays. YEWV and YESV were provided by Dr. Gunge. HYT223 and HYT239 were provided by Dr. Ogawa (Osaka Univ., Japan). Bacteria and yeast cells harboring YEp51-KS were grown in complete medium LB (0.5% yeast extract, 1% polypeptone, 1% NaC1, pH7.2) and selective medium SD (0.67% Difco nitrogen base, 2% glucose, supplemented with the necessary nutrients, pH5.6), respectively. E. coli cells (HB101) as an indicator of mutation were cultured in H medium (1% polypeptone, 0.8% NaCI, pH7.0). The rpsL gene has an amber mutation (TAG) at the sixth codon (CAG), but expresses the wild-type phenotype of streptomycin (Sm) sensitive in the host E. coli HB 101 carrying the amber suppressor supE44 (Gondo et al., 1996). If necessary, kanamycin (Km) and Sm were added at final concentrations of 50 ~tg/ml and 100 ~tg/ml, respectively. Minimal agar plates contained 0.2% (v/v) glucose and 0.008% (v/v) nutrient broth (Difco) in Davis's media. For plating, Difco-agar (2%) was added to the medium.

Table 1. Strain

Genetic type and 10% survival dose of bacteria and yeast strains Gene background 10% survival

X-ray UVC MNU E. coli

KY390 KY396

KT222

Aphr, F-, pro-lac/c~a,A[pro-lac]thi, trpE9777, malE::TnlO KY390 but uvrA6

•(ada-alkB)::kan, F-, thrl, leu6, his4, argE3, proA2, thil, lacY1, galK2, ara14, xyl5, mtll, isx33, rpsL31, supE44

S. cerevisiae YEWV MAT~z/a, leu2, his1, met1 YESV YEWV but rad4 HYT223 a, ura3, leu2, trpl, ade5, his7 HYT239 HYT223 but rad52::hisGURA3hisG

150 Gy 70 Jim 2 150 Gy 2 Jim 2

750 Gy 100 Gy

300 J/m 2 30 J/m 2

100 mM

30 mM

A

B

a b ,~c d

C

Fig. 1. Reaction bag. a, medium; b, a blue bead and membrane filters with dried microorganisms; c, empty room; d, a red bead and stock solution containing glycerol, e, f, g, special temporary sealing band. e, marking with blue arrow; f, marking with red arrow. A, before activation; B, after activation; C, after deactivation.

Effects of Microgravity on Induced Mutation 557

Space Experimental Conditions. Bacteria and yeast cells were grown in medium, and treated with X-rays at 1 Gy/min (150-kVp X-ray generator, Model MBR-1520R, Hitachi, Tokyo, Japan), UVC at 0.1-1 j/m2/s (GL10, Toshiba, Tokyo, Japan) or MNU (Nacalai tesque, Kyoto, Japan) for 1 h at 37°C. The cells carrying damaged-DNA were immediately washed and resuspended in the stabilizing LB medium containing 20 % gelatin and L-buffer (6% polyvinylpyrolidone, 3% sodium glutamate, 5% lactose, 0.1M potassium phosphate, pH7.0), respectively (Mikata and Banno, 1989). Aliquots of about 100 ~tl (2 x 108 cells) from each sample were placed onto individual nitrocellulose sheets (6 mm x 9 ram, ADVANTEC, Tokyo, Japan) and vacuum-dried overnight at 4°C. The medium (500 ~1), membrane filter with dried microorganisms and 550 ~tl stock solution (bacterium samples, 50% glycerol; yeast samples, 30% glycerol) were sealed separately in compartments a, b and d, respectively, in a new apparatus called the reaction bag (1.5 cm x 10.0 cm) (Figure 1A). The reaction bag was specially made of polypropylene sheets (Hybrid MekkinBag HM-1304, HOGY, Osaka, Japan) in our laboratory. The reaction bags were transported from our laboratory at about 4 °C and boarded on the space shuttle 1 day before launch. The space shuttle Discovery (STS-91) was orbiting the earth at an altitude of 400 km and an inclination of 51.6 degrees to the earth' s equator. The space shuttle was launched from the NASA Kennedy Space Center (Florida, USA) on June 2, 1998. To begin culture, the separating filrrks were broken by pushing from one side of the separated compartment in each bag (Figure 1B). The tubes were alternately pushed from one side by the crew's fingers for mixing. In E. coli, the reaction bags were incubated after mixing in the dark at room temperature (21.6-22.1 °C) for 25 h. In S. cerevisiae, we judged the timing of deactivation based on the crew's observations for 2-4 days. During this period, cell growth was advanced under microgravity conditions. In this time, the fixation of induced-mutations was performed in space. The bacteria and yeast cells were stored in a freezer at about -20°C after mixing the culture cells and stock solution containing glycerol (Figure 1C). Using blue and red glass beads (03 mm, No. 5 and 942, TOHO, Osaka, Japan) in each reaction bag, it was easily confirmed that the two kinds of colorless liquids were mixed. Two mission specialists (Franklin R. Chang-Diaz, Ph.D. and Janet Lynn Kavadi, Ph.D.) on board the space shuttle performed each step of activation and deactivation of the cell growth. Control experiments on earth were done at the NASA Johnson Space Center (Houston, USA) at the same temperatures after receiving downlink information on the space experimental conditions from the space shuttle. Measurement of Number of Surviving Cells. The samples were transported to Nara Med. Univ. After thawing, the cells were diluted in part with saline and plated. The number of surviving cells of E. coli and S. cerevisiae per reaction bag was obtained by the method of colony formation. Measurement of Induced Mutation Frequency. For reverse mutation frequencies, the number of revertants from tryptophan dependence (trp) to tryptophan independence (Trp +) (Glickman et al., 1980) or arginine dependence (arg) to arginine independence (Arg +) (Todd et al., 1979) was determined according to the procedure of Kato and Nakano (1981). The forward mutation frequencies were calculated from the fraction of conversion of Sm resistant phenotype of E. coli HBI01 to sensitive phenotype by the transformation with the yeast-bacteria shuttle vector YEp51-KS carrying the E. coli rpsL gene. The wild-type rpsL gene encodes one of the E. coli ribosomal subunit proteins and confers the dominant phenotype of Sm-sensitive on Sin-resistant cells by the transformation. In addition, the plasmid DNA contained a Kin-resistance gene. When a mutation occurs in the rpsL gene, the mutation frequencies are calculated from the ratio of the number of colonies on plates containing Kan and Sm to the number of colonies on plates containing Km (Gondo et al., 1996). Measurement of Induced SOS Response. We determined the induction of expression of umuDC genes that are essential for mutation. We used two E. coli strains, KY390 and KY396, carrying a pSK1002 plasmid. In these systems, it is easy to measure/3-galactosidase activities for the induced activities of SOS-response (Komeda et al., 1997).

RESULTS AND D I S C U S S I O N

We studied the effects of microgravity on the induced-mutation frequencies aboard the S/MM-9 flight. In space, it is difficult to handle and mix microorganisms, culture medium and stock solution to measure exact volumes. Therefore, we developed a new apparatus for space experiments (Figure 1). This reaction bag, made of autoclavable polypropylane sheets, is very low cost, compact, lightweight and easy to mix in space. Using E. coli and S. cerevisiae defective in excision repair, recombinational repair and inducible repair, and their wild type strains, we aimed to clarify the effects of microgravity on each step of repair for mutation. We confirmed the viability of these microorganisms to analyze the effects of microgravity on the induced- mutation frequencies after fixation of mutations in space. The results are shown in figures 2, 3 and 4 for the reverse mutation frequencies, forward mutation frequencies and the induced SOS-responses related to the induction of mutation in E. coli, respectively. The findings obtained from ground control experiments is

558 A. Takahashi et al.

well correspondent with those from previous reports. Although an excision repair deficient the strain, KY396, has almost the same radiosensitivity (Table 1) and radiation-induced mutation frequency as the wild type strain (Figures 2A and 3A), the strain is very sensitive (Table 1) and hypermutable to UVC (Figures 2B and 3B), since uvrA strain is defect to recognize UV induced pyrimidine dimers and to repair by excision repair (Sancar, 1996).

> , o 10-5 t,,-

o" i _

IJ.. lO-S t,.- 0

t~

lO-r

f • i - ,

A i i i

B

I , I L I L , I i I i I

0 50 100 150 0 20 40 60

X-rays (Gy) UVC (J/rn 2)

i

C

i i I I

0 50 100

MNU (mM) A, X-rays; B, UVC; C, alkylating Fig. 2. The effects of microgravity on reverse mutation in E. coil.

agents (MNU). Circle, KY390 (YEp51-KS); triangle, KY396 (YEp51-KS); square, KT222 (YEp51-KS). Open symbol, ground experiment; closed symbol, space experiment. Vertical bars, ±S.D.

Fig. 3.

0 10"z A

O" L .

u. 10.4

.2

10-5 0 50 100 150

X-rays (Gy)

i i i

B

I L

o '

UVC (Jim 2) The effects of microgravity on forward mutation in E. coli.

C

' 5'0 ~ ' 0 1 O0

MNU (mM) A, X-rays; B, UVC; C, alkylating

agents (MNU). Circle, KY390 (YEp51-KS); triangle, KY396 (YEp51-KS); square, KT222 (YEp5I-KS). Open symbol, ground experiment; closed symbol, space experiment. Vertical bars, i- S.D.

10 p.

8 o

X " - " 6 u)

4 "O

O 2' o

m 0

' I ' I '

A

, I , I L

0 100 200

X-rays (Gy)

, I i I ,

0 5 10

UVC (Jim 2)

i

B

15

Fig. 4. The effects ofmicrogravity on SOS-response in E. coli. A, X-rays; B, UVC. Circle, KY390 (pSK1002); triangle, KY396 (pSK1002). Open symbol, ground experiment; closed symbol, space experiment. Vertical bars, ±S.D.

Effects of Microgravity on Induced Mutation 559

It is well known that error-free excision repair may be selectively inhibited, forcing a greater fraction of mutational lesions to be processed by the error-prone component of the post-replication repair system (Webb and Tumer, 1981). Indeed, when the bacterial cells were exposed to UVC, induction of the SOS-response (error-prone) of post-replication repair was higher in the excision-repair-deficient strain than in the wild type strain (Figure 4B). Methylation damage is repairable by diverse DNA-repair pathways including O6-methyl guanine methyl transferase and 3-methyl adenine glycosylase controlled by ada genes (Mitra et al, 1982). We observed an enhancement of cell death after the treatment of MNU, an alkylating agent, in an ada KT222, versus an ada ÷ KY390, strain (Table 1). Alkylating agent-induced mutation frequencies were also higher in an ada strain deficient in alkylated DNA repair systems than the wild type strain (Figures 2C and 3C). The induced mutation frequencies and SOS-responses of the both E. coli strains in space samples were similar to those in the ground control samples (Figures 2, 3 and 4).

1 0 - 2 , , ,

10-3

10-4

o t - o -n ~r Iu

tt

c 0

A

L t i I L t i

0 250 500 750 X-rays (Gy)

i i i

B

, I ~ I L I

0 100 200 300

UVC (Jim 2) Fig. 5. The effects of microgravity on forward mutation in S. cerevisiae. A, X-rays; B, UVC. A, circle, HYT223 (YEp51-KS); triangle, HYT239 (YEp51-KS). B, circle, YWES (YEp51-KS); triangle, YSES

(YEp51-KS). Open symbol, ground experiment; closed symbol, space experiment. Vertical bars, ± S.D.

The findings on forward mutation frequencies obtained from S. cerevisiae are shown in figure 5. In the ground control experiments, higher X-ray-induced mutation frequencies were observed in a recombination- repair deficient rad52 strain, HYT239, than in the wild type strain, HYT223 (Figure 5A). It is well known that X-rays produce mainly DNA strand breaks that can be repaired by homologous recombination repair with rad52 (Resnick and Martin, 1976). Therefore, rad52 deficient strain is very sensitive to ionizing radiation (Table 1) and is mutable (Figure 5) compared with the wild type strain. Rad4 is involved in excision repair (Prakash, 1977). The UV sensitivity of the yeast strain defecting rad4 was similar to that of E. coli uvrA deficient strain. UV-induced mutation frequency was higher in the rad4 strain than in the wild-type strain (Figure 5B). The findings clearly show that there is no significant difference in forward mutation frequencies between the space samples and the ground control samples in S. cerevisiae (Figure 5). We obtained the results that microgravity does not alter the frequency of induced mutation and the level of induction of SOS response. The results might lead to the assumption that microgravity also does not affect DNA replication and DNA repair (excision repair, recombinational repair and inducible repair). We also found no significant effects of microgravity on the ligation of damaged DNA during space flight using in vitro systems (Takahashi et al., 2000). These findings support those of previous space experiments (Harada et al., 1997; Ohnishi et al., 1997; Takahashi et al., 1997; Pross and Kiefer, 1999; Horneck et al., 1996). Essential mechanism to recognize and to repair damaged DNA is thought to be similar in all organisms on the earth (Eisen and Hanawalt, 1999). We used two different species, prokaryotic bacteria and eukaryotic yeast, to test whether response to microgravity is different between both organisms. In future studies, the effects of microgravity on mutation induced by space radiation should be studied to provide data useful for determining how to protect crew health during prolonged stays in space from carcinogenesis. Furthermore, to clarify microgravity effects in space, researchers will be required to perform experiments under microgravity and lg in space laboratories. The present findings may allow us to determine the RBE of space radiation without consideration of microgravity. The maximum permissible stay in space with respect to the crews' health might be determined from the calculation of RBE of space radiation. We hope that the present data will provide useful information in developing physical protection from the serious effects of space radiation during prolonged space travel.

560 A. Takahashi et al.

ACKNOWLEDGMENTS

The successful completion of space experiments is only possible with the contribution of many people. 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.

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