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Carcinogenesis, Vol. 24, No. 5, 911-917, May 2003
© 2003 Oxford University Press

MOLECULAR EPIDEMIOLOGY AND CANCER PREVENTION

Mutagenic events induced by 4-hydroxyequilin in supF shuttle vector plasmid propagated in human cells

Manabu Yasui, Saburo Matsui, Y.R.Santosh Laxmi1, Naomi Suzuki1, Sung Yeon Kim1, Shinya Shibutani1 and Tomonari Matsuda2

Department of Technology and Ecology, Graduate School of Global Environmental Studies, Kyoto University, Sakyo-ku Yoshida-honmachi, Kyoto, 606-8501, Japan
1 Laboratory of Chemical Biology, Department of Pharmacological Sciences, State University of New York, Stony Brook, NY 11794-8651, USA

2 To whom correspondence should be addressed Email: matsuda{at}eden.env.kyoto-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Increased incidence of breast, ovarian and endometrial cancers are observed in women receiving estrogen replacement therapy (ERT). Equilin and equilenin are the major components of the widely prescribed drug used for ERT. These equine estrogens are metabolized primarily to 4-hydroxyequilin (4-OHEQ) and 4-hydroxyequilenin, respectively, which are autoxidized to react with DNA, resulting in the various DNA damages. To explore the mutagenic potential of equine estrogen metabolites, a double-stranded pMY189 shuttle vector carrying a bacteria suppressor tRNA gene, supF, was exposed to 4-OHEQ and transfected into human fibroblast. Plasmids containing mutations in the supF gene were detected with indicator bacteria and mutated colonies obtained were analyzed by automatic DNA sequencing. The proportion of plasmids with the mutated supF gene was increased dose-dependently. The majority of the 4-OHEQ-induced mutations were base substitutions (78%); another 22% were deletions and insertions. Among the base substitutions, 56% were single base substitutions and 19% were multiple base substitutions. The majority (86%) of the 4-OHEQ-induced single base substitutions occurred at the C:G site. C:G -> G:C and C:G -> A:T mutations were detected preferentially with lesser numbers of C:G -> T:A transitions. Sixty-two percent of base substitutions were observed particularly at C:G pairs in 5'-TC/AG-5' sequences. Using 32P-post-labeling/gel electrophoresis analysis, 4-OHEN–dC was a major adduct, followed by lesser amounts of 4-OHEN–dA adduct. Mutations observed at C:G pairs may result from 4-OHEN–dC adduct. These results indicated that 4-OHEQ is mutagenic, generating mutations primarily at C:G pairs in 5'-TC/AG-5' sequences.

Abbreviations: ERT, estrogen replacement therapy; HPLC, high-performance liquid chromatography; 4-OHEQ, 4-hydroxyequilin; 4-OHEN, 4-hydroxyequilenin; 8-oxodG, 8-oxo-7,8-dihydro-2'-deoxyguanosine; 8-oxodA, 8-oxo-7,8-dihydro-2'-deoxyadenosine; PAGE, polyacrylamide gel electrophoresis.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Estrogen replacement therapy (ERT) is most widely used among postmenopausal women to decrease menopausal symptoms and also to protect against osteoporosis (1). More than 40% of US women in this group currently receive ERT. Premarin (Wyeth-Ayerst), composing of ~50% estrogen, 30% equilin, 10% equilenin and 10% 8,9-dehydroestrogen, is used for this purpose (reviewed in ref. 2). However, ERT showed a significant increase in the risk of developing breast (3,4), ovarian (5) and endometrial cancers (6). Significant elevation of cancer risks was associated with increasing duration of ERT (5,7). The occurrence of endometrial hyperplasia was observed in postmenopausal women receiving ERT (8). Treatment of hamsters for 9 months with estrone, equilin or equilenin resulted in 100% tumor incidence and many tumor foci in kidneys (9). DNA adducts have also been detected in the tissues of rodents treated with natural and synthetic estrogens (10). Major metabolites of equine estrogens react readily with the DNA in vitro, resulting in the formation of large number of DNA adducts (2,11). Therefore, exogenous estrogens may be involved in the initiation of breast, ovarian and endometrial cancers.

Like estrogen, equilin and equilenin are metabolized to 4-hydroxyl and 2-hydroxyl forms (2). However, there is no information regarding the level of equine estrogens and their fate in tissues of women treated with Premarin although pharmacokinetics of Premarin has been reported (12,13). 4-Hydroxyequilenin (4-OHEN) is rapidly autoxidized to an o-quinone which in turn readily reacts with DNA, resulting in the formation of unique dC, dA and dG adducts (Figures 1 and 2); the dA adduct is unstable, generating apurinic sites (14). Using 32P-post-labeling/polyacrylamide gel electrophoresis (PAGE) analysis (15), we have observed that 4-OHEN are highly reactive with DNA; large amounts of 4-OHEN–dC adduct, accompanied by lesser amounts of 4-OHEN–dA adduct, are detected. 4-Hydroxyequilin (4-OHEQ) is also autoxidized to an o-quinone, which isomerizes to 4-OHEN o-quinone (Figure 1); therefore, 4-OHEQ produces identical DNA adducts that are observed with 4-OHEN (16). During redox cycling between 4-OHEN o-quinone and their semi-quinone radicals, it generates superoxide, hydrogen peroxide and ultimately reactive hydroxyl radicals (17). When 4-OHEN was incubated with DNA in vitro or exposed to cultured breast cancer cells, increased formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydro-2'-deoxyadenosine (8-oxo-dA) adducts was observed in the DNA (17–21). Particularly, when 4-OHEN was injected directly into the mammary fat pads of rats, 4-OHEN–dA and 4-OHEN–dG adducts, in addition to increased formation of 8-oxodG, were detected in the mammary tissue using LC/MS/MS spectroscopy (22). If equine estrogen–DNA adducts are mutagenic, such DNA adducts may have association in initiating cancers.



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  		Fig. 1. Mechanism of formation of 4-OHEN-derived DNA adducts.

 


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  		Fig. 2. Structures of DNA adducts induced by 4-OHEN or 4-OHEQ.

 
In the present study, pMY189 shuttle vector plasmids were exposed to 4-OHEQ and transfected into human fibroblast. The mutational frequency and spectrum occurred in the supF gene of the plasmids were analyzed. We found that 4-OHEQ, an equine estrogen metabolite, was mutagenic.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Materials
Ampicillin, chloramphenicol, nalidixic acid, isopropyl-ß-D-thiogalactoside (IPTG) and 5-bromo-4-chloro-3-indoyl-ß-D-galactoside (X-gal) were obtained from Nakalai Tesque (Kyoto, Japan). Calf thymus DNA, micrococcal nuclease and potato apyrase were purchased from Sigma-Aldrich (St Louis, MO). Spleen phosphodiesterase was obtained from Worthington Biochemical (Freehold, NJ). 3'-Phosphatase free T4 polynucleotide kinase was purchased from Roche Boehringer Mannheim (Indianapolis, IN). Restriction endonuclease DpnI was obtained from TOYOBO (Shiga, Japan). LIPOFECTAMINETM Reagent was purchased from Gibco BRL (Gaithersburg, MD). QIAGEN Plasmid-kit and QIAprep-spin Plasmid kit were purchased from Qiagen GmbH (Hilden, Germany). [{gamma}-32P]ATP (sp. act. >6000 Ci/mmol) was obtained from Amersham Pharmacia Biotech (Piscataway, NJ).

Human cells
A SV40-transformed normal human fibroblast cell line WI38-VA13 (21) obtained from American Type Culture Collection (Rockville, MD) was used in this study. The cells were cultured in Dulbecco's modified minimum essential medium (Nikken, Kyoto, Japan) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT) at 37°C in a CO2 incubator.

Shuttle vector plasmid and bacterial strains
The shuttle vector plasmid pMY189, constructed previously by Matsuda et al. (24), was used for analysis of mutations induced by 4-OHEQ. The pMY189 was derived from the pZ189 (25) as described previously (24). The indicator Escherichia coli strain KS40/pKY241 (26), which is a nalidixic acid-resistant (gyrA) derivative of MBM7070 [lacZ(am) CA7070 lacY1 HsdR HsdM {Delta} (araABC-leu) 7679 galU galK rpsL thi] (27), was used for detection of the mutated pMY189. The plasmid pKY241 was constructed by Akasaka et al. (26) and contained a chloramphenicol resistant marker and a gyrA (amber) gene.

Transfection of pMY189 modified with 4-OHEQ to human cells
4-OHEQ was prepared using an established protocol (28). A purified stock of pMY189 was prepared with the QIAGEN Plasmid Purification Kit. The plasmid pMY189 (20 µg) was reacted at 37°C for 1 h with variable amounts of 4-OHEQ (0.02–2.0 mg in 10 µl DMSO) in 1.0 ml of 25 mM potassium phosphate buffer (pH 7.4). After the reaction, the plasmid DNA was recovered by ethanol precipitation, evaporated to dryness and then dissolved in TE buffer (pH 8). The human WI38-VA13 cells (2 x 107) were transfected with 4-OHEQ-treated pMY189 (2 µg) with LIPOFECTAMINETM Reagent and incubated at 37°C for 72 h in a CO2 incubator. Then, the plasmid was extracted from the cells using QIAprep-spin Plasmid kit and digested with the restriction endonuclease DpnI to eliminate non-replicated input plasmids with the bacterial methylation pattern.

Analysis of mutated supF gene
Plasmid DNA was introduced into the indicator bacteria E.coli KS40/pKY241 by the electroporation apparatus E.coli Pulser (Bio-Rad Laboratories, Hercules, CA). The bacteria were spread on LB agar plates containing 50 µg/ml of nalidixic acid, 150 µg/ml of ampicillin and 30 µg/ml of chloramphenicol, together with IPTG and X-gal. Plasmids with mutated supF genes made E.coli cells resistant to nalidixic acid, whereas cells carrying plasmids with unmutated supF genes could not grow in the presence of nalidixic acid. IPTG and X-gal were added to confirm selection of the mutated supF gene by the color of the colonies. A portion of the bacteria was spread on plates containing ampicillin and chloramphenicol to measure the transformant fraction and plasmid survival. Mutated plasmids were extracted and purified from the overnight culture. The sequences of the supF gene of the plasmids were determined with the -21M13 primer and Dye-Primer Cycle Sequencing reagent kit using a 370A automatic DNA sequencer (Applied Biosystems Foster, CA).

Determination of 4-OHEQ-derived DNA adducts in pMY189 plasmid
Plasmid DNA sample (0.1 µg) was enzymatically digested at 37°C for 2 h in 30 µl of 17 mM sodium succinate buffer (pH 6.0) containing 8 mM CaCl2, using micrococcal nuclease (15 U) and spleen phosphodiesterase (0.05 U). The reaction mixture was incubated for another 1 h with nuclease P1 (1.0 U). The DNA digests were incubated at 37°C for 40 min with [{gamma}-32P]ATP (20 µCi) and 3'-phosphatase free T4 polynucleotide kinase (20 U) in 20 µl of 500 mM Tris–HCl buffer (pH 7.5) containing 100 mM MgCl2, 100 mM DTT and 10 mM spermidine, and then incubated with 50 mU of apyrase for another 30 min. 4-OHEQ (2 mg in 10 µl DMSO) was also reacted at 37°C for 5 h with dN3'P (0.5 mg) in 1.0 ml of 25 mM potassium phosphate buffer, pH 7.4. After the centrifugation, one-twentieth of the supernatant was evaporated to dryness and incubated at 37°C for 1 h with nuclease P1 (1.0 U) in 20 µl of 17 mM sodium succinate buffer (pH 6.0) containing 8 mM CaCl2. The reaction mixture was evaporated to dryness and labeled with 32P, as described for the 4-OHEQ-modified DNA samples. A part of the 32P-labeled samples was analyzed by 32P-post-labeling/PAGE (15). 4-OHEN was synthesized using an established protocol (14) and reacted with dC3'P and dA3'P, as described for 4-OHEQ. 4-OHEN–dC3'P and 4-OHEN–dA3'P were isolated by HPLC (29) and their molecular weight was confirmed using FAB mass spectroscopy. 4-OHEN–dC3'P and 4-OHEN–dA3'P were used as standards for analysis of 4-OHEQ-derived DNA adducts.

32P-Post-labeling/PAGE analysis
A part of the 32P-labeled sample was electrophoresed for 4–5 h on a non-denaturing 30% polyacrylamide gel (35 x 42 x 0.04 cm) with 1400–1600 V/20–40 mA (15). A 30% polyacrylamide gel was prepared from 40% polyacrylamide solution (60 ml), 10x TBE buffer, pH 7.0 (10 ml), distilled water (10 ml), 10% ammonium persulfate (0.6 ml) and TEMED (35 µl). Ten times TBE buffer (pH 7.0) was prepared from 1 M Tris-base, 2.24 M boric acid and 25.5 mM EDTA. The position of 32P-labeled adducts was established by a ß-PhosphorImager analysis (Molecular Dynamics, Sunnyvale, CA). To determine the radioactivity of 32P-labeled products, integrated values were measured using a ß-PhosphorImager. Values ranging from 1 to 108 had a linear response (data not shown). When the radioactivity was beyond the range, the shorter exposure to 32P-labeled products was used to determine the radioactivity within the linear range. The relative adduct levels were calculated according to Levay et al. (30): for example, (total d.p.m. in adducts)/5.30 x 109 d.p.m., assuming that 0.1 µg of DNA was 3.03 x 102 pmol of dN3'P and the specific activity of the [{gamma}-32P]ATP was 1.75 x 107 d.p.m./pmol. The specific activity of the [{gamma}-32P]ATP was corrected by calculating the extent of decay. When known amounts of an oligodeoxynucleotide containing a single 4-OHEN–dC adduct was mixed with calf thymus DNA and used as a standard for 32P-postlabeling analysis, the recovery of 4-OHEN–dC adduct was 50% (S.Shibutani, unpublished data). Therefore, the actual level of 4-OHEN–dC adduct was estimated by dividing the experimental values by 50%. Although the recovery of 4-OHEN–dA has not been determined using the site-specific modified oligomer, the same recovery rate was applied to estimate the level of 4-OHEN–dA adduct.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Level of 4-OHEQ-derived DNA adducts in plasmid vectors
When dN3'P was reacted with 4-OHEQ under neutral buffer conditions and labeled with 32P, 4-OHEQ-modified dA3'P, dC3'P and dG3'P were detected by the 32P-post-labeling/PAGE analysis (Figure 3). No adducts were detected with dT3'P. Using 32P-post-labeling/PAGE analysis, the level of 4-OHEQ-derived DNA adducts in the plasmid vectors which were reacted with variable amounts (0.02–2.0 mg) of this compound were analyzed. As reported earlier (16), it was observed that 4-OHEQ was auto-oxidized to form 4-OHEQ o-quinone, which was isomerized to form 4-OHEN o-quinone. Therefore, 4-OHEQ o-quinone reacts with the DNA, resulting in the identical DNA adducts formed by 4-OHEN o-quinone. As shown in Figure 3, 4-OHEN–dC adduct, followed by 4-OHEN–dA adduct, was detected as a major adduct. The formation of 4-OHEN–dC and 4-ONEN–dA adducts increased dose-dependently (Figure 4). When the plasmid (20 µg) was reacted with 0.5 mg (final 1.8 mM concentration) of 4-OHEQ, the level of 4-OHEN–dC and 4-OHEN–dA adducts were 5.8 adducts/104 dN and 0.06 adducts/104 dN, respectively. Several unknown adducts were also detected as minor products. No adducts were detected in the control DNA sample.



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  		Fig. 3. Determination of DNA adducts derived from 4-OHEQ. pMY189 plasmid (20 µg) was reacted at 37°C for 1 h with 0.02–2.0 mg of 4-OHEQ in 1.0 ml of 25 mM potassium phosphate buffer, pH 7.4. The recovered DNA (0.1 µg) was digested using nuclease P1 enrichment method and labeled with 32P, as described in the Materials and methods. 4-OHEQ (2 mg) was also incubated at 37°C for 5 h with 0.5 mg of dA3'P, dC3'P, dG3'P or dT3'P in 1.0 ml of 25 mM potassium phosphate buffer, pH 7.4. The dN3'P samples (25 µg) was incubated for 1 h with nuclease P1 (1.0 U) in 30 µl of 17 mM sodium succinate buffer (pH 6.0) containing 8 mM CaCl2. The reaction mixture was evaporated to dryness and labeled with 32P. A one-fifth volume of the 32P-labeled samples was developed for 5 h on a 30% polyacrylamide gel electrophoresis. 4-OHEN–dC3'P and 4-OHEN–dA3'P were also labeled with 32P, as described for 4-OHEQ-derived dN3'P and used as standards. [{gamma}-32P]ATP (16.7 fmol) was subjected to the gel to quantify DNA adducts derived from 4-OHEQ.

 


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  		Fig. 4. Dose–response curve of 4-OHEN–dC and 4-OHEN–dA formation. Data were taken from Figure 3.

 
Mutations induced by 4-OHEQ on supF gene
4-OHEQ-treated vector plasmid pMY189 was transfected into the human WI38-VA13 cells and incubated for 3 days to allow replication and mutation fixation. The progeny plasmids were recovered. An indicator E.coli was transformed by the plasmids to determine the plasmid survival and mutation frequency. The number of ampicillin-resistant bacterial colonies was reduced by the increased exposure of 4-OHEQ to the shuttle vectors; in contrast, the proportion of plasmids with the mutated supF gene was increased (Figure 5). When plasmids were treated with 1.8 mM of 4-OHEQ, the mutation frequency (28 x 10-4) was 12-times higher than that of the control (2.3 x 10-4).



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  		Fig. 5. Survival of 4-OHEQ-treated pMY189 plasmids propagated WI38-VA13 (left panel) and mutation frequency of 4-OHEQ-treated pMY189 plasmids propagated WI38-VA13 (right panel). The ratio of the number of ampicillin-resistant bacterial colonies with 4-OHEQ-treated plasmids to the number with the untreated plasmids is shown as plasmid survival. The frequency of bacterial colonies with the mutated supF gene in all the ampicillin-resistant bacterial colonies is shown as mutation frequency. Average numbers of three independent experiments are plotted with the SD of the mean.

 
Seventy-eight mutant colonies were obtained from the plasmid treated with 1.8 mM of 4-OHEQ and subjected to sequence analysis. The majority of the 4-OHEQ-induced mutations were base substitutions (78%); the remaining 22% were deletions and insertions (Table I). Among the base substitutions, 56% showed single base substitutions and 19% showed multiple base substitutions (at least two base substitutions except for the tandem base substitutions). Distribution of the base substitutions observed in the supF gene is shown in Figure 6. The majority of the 4-OHEQ-induced single base substitutions occurred at the C:G sites (86%). C:G -> G:C (48%) and C:G -> A:T (28%) transversions were observed primarily, along with lesser number of C:G -> T:A transitions (10%) (Table II). Small number of A:T -> T:A transversions (8%) and A:T -> G:C transitions (8%) were also observed.


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  		Table I. Types of mutations in the supF gene in 4-OHEQ-treated shuttle vector plasmid pMY189

 


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  		Fig. 6. Distribution of 4-OHEQ-induced base substitutions in supF gene. The asterisks represent C:G pairs in 5'-TC/AG-5' sequences. Underlined are tandem and ternate base substitutions. Multiple mutations, deletions, and insertions are as follows (the site of the change is shown in parentheses). G -> C (70), G -> A (105) and G -> T (111); G -> C (70), G -> A (105) and G -> T (111); G -> C (70) and G -> T (111); C -> A (108) and C -> A (163); C -> A (133) and A -> T (137); C -> A (149), C -> A (155) and C -> A (168); G -> C (65), G -> C (111), G -> A (150) and C -> T (178); T -> C (50), G -> A (65) and G -> C (126); C -> A (108) and C -> A (163); C -> G (118), C -> A (139), C -> A (149) and C -> T (185); C -> G (133) and C-> T (185); C -> G (133) and C -> T (146); G -> A (156) and C -> A (179); G -> A (129) and C -> A (133); deletion (135); deletion (131–154); deletion (127–129); deletion (129–155); deletion (46–61); deletion (94–111); deletion (117–129); deletion (128–136); deletion (146–176); deletion (117–197); large deletion (98–); large deletion (178–); large deletion (85–); large deletion (174–); C insertion (123–124).

 

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  		Table II. Types of 4-OHEQ-induced single base substitutions in the supF gene

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Mutagenic potential of 4-OHEQ was determined using human fibroblast and pMY189 shuttle vector carrying supF gene exposed to 4-OHEQ. 4-OHEQ promoted base substitutions preferentially at C:G sites. There are 21 5'-TC and 5'-GA sequences in the target site of supF gene. Sixty-two percent of base substitutions occurred at C:G pairs in 18 of 21 5'-TC/AG-5' sequences. This suggests that C:G pairs in the 5'-TC/AG-5' sequences are the major targets damaged by 4-OHEQ and results in large numbers of base substitutions. Further studies are required in this direction of the chemical preference of 4-OHEQ at this 5'-TC/AG-5' sequence.

As shown in Table II, C:G -> G:C and C:G -> A:T transversions were detected primarily, along with lesser numbers of C:G -> T:A transitions. These mutations may be promoted from C and/or G damaged by 4-OHEQ. Bolton and her colleagues have reported that 4-OHEN reacts with DNA, resulting in the formation of 4-OHEN–dC, 4-OHEN–dA and 4-OHEN–dG adducts (14). However, when pMY189 plasmid exposed to 4-OHEQ was analyzed using 32P-postlabeling/PAGE, 4-OHEN–dC adduct was the major adduct, followed by lesser amounts of 4-OHEN–dA adduct (Figures 3 and 4). Similar observations were seen for the DNA reacted with 4-OHEN (15). Therefore, mutations detected at C:G pairs in supF gene may have resulted from 4-OHEN–dC adducts.

The mutant frequency was plotted against the number of DNA adducts (4-OHEN–dC plus 4-OHEN–dA) per mutational target sites (Figure 7). The supF gene consisted of 150 bp nucleotides, but not all the bases are the targets. Literature review revealed that there are 93 target bases in supF gene (24). From the regression curve of Figure 6, it was estimated that 38 4-OHEN–dC or 4-OHEN–dA adducts will produce one mutation.



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  		Fig. 7. Relationship between 4-OHEQ DNA adducts and mutant frequency.

 
The other possible DNA damages are due to the oxidative adducts induced by 4-OHEQ. 4-OHEQ is autoxidized to an o-quinone, which isomerizes to 4-OHEN o-quinone (16). Reactive oxygen species, which are generated during redox cycling between 4-OHEN o-quinone and their semiquinone radicals, react with cellular DNA, resulting in an increased formation of 8-oxodG and 8-oxodA adducts both in vitro and in vivo (17–21). 8-oxodG has been known to create mutagenic lesions, primarily generating G -> T transversions in mammalian cells (31–33). 8-oxodG generated in C:G pairs might contribute in part to the C:G -> A:T mutations observed with 4-OHEQ.

Small numbers of A:T -> T:A transversions and A:T -> G:C transitions were also detected at A:T pairs in supF gene. One of the authors (S.S.) has reported that 8-oxodA is relatively a weak mutagenic lesion, generating A -> C transversions in mammalian cells (33). Since this mutagenic spectrum was not detected in the present study, contribution of 8-oxodA adduct to mutations generated by 4-OHEQ might be minimal. Mutations observed in A:T pairs might be promoted by 4-OHEN–dA adducts. Therefore, mutations observed in supF gene may be due to the covalent dC and dA adducts and/or 8-oxodG generated by 4-OHEQ.

In conclusion, we have found that 4-OHEQ, a major metabolite of equilin, is mutagenic, generating primary C:G -> G:C and C:G -> A:T transversions; sequence dependent mutations were also observed at C:G pairs in 5'-TC/AG-5' sequences. Equilin was thought to be a cancer promoter via the activation of the estrogen receptor in the target tissues. However, 4-OHEQ was found to damage DNA and cause mutations, indicating that equilin might act as an initiator of developing breast, ovarian and endometrial cancers.


   Acknowledgments
 
This research was supported by the National Institute of Environmental Health Sciences Grant ES09418, Grants-in-aid for Scientific Research 13027245 from the Ministry of Education, Culture, Sports, Science and Technology (MEXT, Japan) and New Energy and Industrial Technology Development Organization (NEDO, Japan).


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received December 9, 2002; revised February 6, 2003; accepted February 17, 2003.


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