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Antimutagenic factors in aquatic plants
Mutation Research, 178 (1987) 211-216
211
Elsevier MTR 04353
Antimutagenic factors in aquatic plants Takako Fujimoto, Youki Ose, Takahiko Sato, Hiroaki Matsuda, Hisamitsu Nagase and Hideaki Kito
Gifu PharmaceuticalUniversity,5-6-1Mitahora-higashi, GifuCity, Gifu(Japan) (Received 31 July 1986) (Revision received 24 December 1986) (Accepted 6 January 1987)
Keywords: Aquatic plants; Antimutagenic factors; Curled pondweed; European cut-grass; Smartweed. Summary Antimutagenic activity was screened in water extracts from 13 kinds of aquatic plant and it was found that curled pondweed (Potamogeton crispus L.), European cut-grass (Leersia japonica Makino) and smartweed (Polygonum hydropiper L.) had large antimutagenic effects. Antimutagenic factors in these extracts were heat-resistant. The factors in curled pondweed and European cut-grass reduced the mutagenicities of benzo[a]pyrene and 2-nitrofluorene, but did not reduce the mutagenicity of AF-2. The factor in smartweed reduced the mutagenicity of benzo[a]pyrene, but did not reduce the mutagenicity of 2-nitrofluorene and AF-2. The factor in curled pondweed had molecular weight (mol. wt.) above 300000. The factors in European cut-grass and smartweed existed in both fractions with mol. wt. above and below 300 000. The active factors in curled pondweed and smartweed may act as the desmutagen, but that in European cut-grass does not act as a desmutagen.
The authors reported that a kind of aquatic plant, grass-wrack pondweed (Potamogeton oxyphyllus Miq.) had a desmutagenic effect and the factor was heat-resistant and had a mol. wt. above 300000 (Sato et al., 1984). In this paper, the authors collected 13 aquatic plants from some tributaries of Nagara River in Gifu Prefecture and the antimutagenic effect on benzo[a]pyrene was screened and it was found that some plants had the effect. The nature of these antimutagenic factors was investigated.
Correspondence: Dr. Takahiko Sato, Associate Professor, Department of Environmental Hygiene, Gifu Pharmaceutical University, 5-6-1 Mitahora-higashi, Gifu 502 (Japan).
Materials and methods
(1) Sampling of plants Aquatic plants were collected from some tributaries of Nagara River, such as Toba, Fukutomi and Arata Rivers in Gifu Prefecture.
(2) Preparation of water extracts Plants were washed with tap and distilled waters, minced with scissors, ground with mortar and pestle, and homogenized by a mixer with distilled water. Homogenates were centrifuged at 3500 g for 20 min and supernatants were filtered through a gauze. The filtrates were concentrated by a rotary evaporator and freeze-dried. After
0027-5107/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
212
being dissolved in proper distilled water, they were used for the experiments. For sterilization, centrifugation (1100 g, 30 min) was used at first, but later an autoclave was used, because it was known that these antimutagenic factors were heat-resistant.
(3) Test of antimutagenic effect on mutagens The antimutagenic effects of these extracts on mutagens were investigated with Salmonella typhimurium TA100 and TA98 as described by Sato et al. (1984). If metabolic activation was needed, $9 mix was added. $9 fraction was prepared from male Sprague-Dawley rats treated
with PCB (Kanechlor-400). After dilution with M / 1 5 phosphate buffer (pH 7.0), the number of surviving cells was determined by B-2 medium (Kada, 1973).
(4) Heat effect on the antimutagenic factors After sterilization by centrifugation, half of the supernatant was autoclaved at 121°C for 20 min. The antimutagenic effect on benzo[a]pyrene was investigated with the heat-treated and untreated supernatants. TA100 and 10 # g / p l a t e of benzo[a]pyrene were used and $9 mix was added. The amounts of extracts were 3 and 6 mg/plate.
TABLE 1 A N T I M U T A G E N I C E F F E C T S OF A Q U A T I C P L A N T E X T R A C T S ON B E N Z O [ a ] P Y R E N E Common name
Scientific n a m e
Smartweed
Polygonum hydropiper L.
A m o u n t of extract (mg)
Antimutagenic activity
8
-0.2 9.2
4
Curled pondweed
Potamogeton crispus L.
European cut-grass
Leersia japonica Makino
Eurasian water milfoil
Myriophyllum spicatum L.
8 4
5.6 48.2
8 4
23.1 61.0
8 4
28.8 52.2
Crowfoot
Ranunculus mpponicum Nakai var. submersus Hara
8 4
40.4 62.8
Ukishiba
Chamaeraphis spinescens Poir. var. depauperata Hook. fit.
8 4
50.2 32.2
Kougaimo
Vallisneria denseserrulata Makino
8 4
54.6 88.2
Small pondweed
Potamogeton pusillus L.
8 4
58.7 80.5
Eelgrass
Vallisneria natans Hara
8 4
59.7 69.2
Waterwort
Elatine triandra Schk. var. pedicellata Krylov
8 4
66.3 87.3
Knotweed
Polygonum thunbergii Sieb. et Zucc.
8 4
69.5 92.8
Florida elodea
Elodea nuttalli ( Flanch.) St. John
8 4
110.8 110.5
8 4
203.8 110.6
Water thyme
Hydrilla varticillata Casp.
TA100 ( + $9 mix) and 10 # g / p l a t e of benzo[a]pyrene were used.
213 TABLE 2
(6) Desmutagen test
HEAT EFFECT ON THE ANTIMUTAGENIC FACTORS
The extracts were dissolved to 30 m g / m l of water and sterilized. 2 or 4 ml of the solutions and 200 /~g of b e n z o [ a ] p y r e n e were mixed and incubated at 3 7 ° C for I h with slow shaking. Benzo[a]pyrene was extracted 3 times b y 5 ml of ethyl acetate, and the ethyl acetate was evaporated to dryness. The sample was redissolved in 2 ml of D M S O , and 0.1 ml of it was supplied to the A m e s test. It was determined beforehand that the antimutagenic substances of these plants were not extractable with ethyl acetate.
His + revertants per 10 6 survivors Curled European Smartweed pondweed cut-grass Spontaneous mutation Benzo[a]pyrene only
6.0 17.4
8.1 27.5
7.1 32.9
3 mg of extract/plate non-autoclaved autoclaved
10.1 9.6
17.3 18.0
14.4 13.9
6 mg of extract/plate non-autoclaved autoclaved
7.3 8.0
13.5 13.4
9.3 9.6
Results
(1) Screening of antimutagenic effect of water extracts on benzo[a]pyrene (5) Antimutagenic effect of fractions fractioned by moL wt. The extracts were fractionated into 2 fractions, that is, "mol. wt. above 300 000 and below 300 000 using ultrafiltration (Diaflo membrane, A m i c o n XM300). Each fraction was freeze-dried and used for the experiment. TA100 and 10 # g / p l a t e of b e n z o [ a ] p y r e n e were used and $9 mix was added. The a m o u n t of samples was 3 m g / p l a t e .
13 aquatic plants were investigated to find the antimutagenic effect on benzo[a]pyrene. F o r indication of antimutagenic effect, the antimutagenic activity was calculated by the following equation. Antimutagenic activity = (S
N)
-
xlO0
(P-N)
S, n u m b e r of revertants when the sample and
Curled pondweed
q 15
~
European c u t - g r a s s
D
5
Smartweed
lOO
5
25
50
~C .
>
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
n-
A m o u n t of e x t r a c t ( m g / p l a t e )
Fig. 1. Dose-response relationship between the amount of extracts and the mutagenicity of benzo[a]pyrene. TA100 (+ $9 mix) and 10/~g/plate of benzo[a]pyrene were used. Broken line indicates spontaneous revertants.
214
Smartweed Curled pondweed
40 w
100 >0
-
>
•
•
•
I European cut-grass
v
40
m
o
30 ~
[ •
v
I
30
•
•
•
20
4o
2t 10
g 2o I
I
I
I
2
4
6
8
10
•
..... ; ......
......
i .......
/
I
2 4 6 8 Amount of extract (mg/plote)
2
I
I
I
4
6
8
Fig. 2. D o s e - r e s p o n s e relationship between the a m o u n t of extracts and the mutagenicity of AF-2. TA100 ( + $9 mix) and 0.05 /~g/plate of AF-2 were used. Broken line indicates spontaneous revertants.
smartweed (Polygonum hydropiper L.) showed strong antimutagenic effect on benzo[a]pyrene. The properties of the antimutagenic factors were further investigated.
benzo[a]pyrene were added to TA100 simultaneously; N, number of revertants by spontaneous mutation; P, number of revertants by benzo[a]pyrene. The results are shown in Table 1. Curled pondweed (Potamogeton crispus L.), European cut-grass (Leersiajaponica Makino) and
(2) Effect of heating on the antimutagenic factors The heat resistance on antimutagenic factors in
Curled pondweed European cut-gross 40(: 300 O >
300
> o3
t ~"t• 10•"
S m o r t w e e d
q
3C !0
•
•
200
% 200
20
Q.
100 c. 100
10
i
i
i
I
2
4
6
8
I
I
I
i
2 4 6 8 Amount of extract (rag/plate)
......
I ......
1 ......
1 ......
1 ....
2
4
6
8
Fig. 3. D o s e - r e s p o n s e relationship between the a m o u n t of extracts and the mutagenicity of 2-nitrofluorene. TA100 ( + $9 mix) and 4 # g / p l a t e of 2-nitrofluorene were used. Broken line indicates spontaneous revertants.
215 TABLE 3 A N T I M U T A G E N I C E F F E C T OF T H E F R A C T I O N S W I T H mol. wt. ABOVE A N D B E L O W 3130000 ON P E N Z O [ a ] P Y R E N E His + revertants per 106 survivors
Spontaneous mutation Benzo[ a ]pyrene only Unfiltrated sample Fraction with mol. wt. above 300000 Fraction with mol. wt. below 3130000
Curled pondweed
European cut-grass
Smartweed
13.3 47.8 12.2 24.7 46.6
13.3 47.8 21.0 28.8 27.3
14.0 63.9 31.5 26.9 25.1
the extracts was examined and the results are shown in Table 2. To ascertain that the decrease of revertants did not depend on a decrease in the number of surviving cells, the number of surviving cells was determined and the frequency of revertants in survivors was calculated. In some cases, a little decrease of surviving cells numbers was observed, especially in the case of mutagens and no extract addition. There are no differences between His + revertants in survivors in 3 non-autoclaved samples and those in 3 autoclaved samples and it is clear that these antimutagenic factors are heat-resistant.
with tool. wt. above 300 000 decreased the mutagenicity of benzo[a]pyrene to about a half, but the fraction with mol. wt. below 300 000 had no effect and it can be considered that the tool. wt. of the antimutagenic factor in curled pondweed was above 300000. While in the case of European cut-grass and smartweed, the fractions with mol. wt. above and below 300 000 decreased the mutagenicity of benzo[a]pyrene to about a half and it can be considered that the mol. wt. of the antimutagenic factors in these plants is contained in fractions with mol. wt. both above and below 300 000.
(5) Desmutagen test (3) Antimutagenic effect of the extracts on various mutagens The authors examined the antimutagenic effect of the extracts on various mutagens, such as benzo[a]pyrene, 2-nitrofluorene and AF-2. The results are shown in Figs. 1, 2 and 3. The extracts of 3 plants had an antimutagenic effect on benzo[a]pyrene, but had not on AF-2. For 2-nitrofluorene, the effect differed between the three plants, that is, curled pondweed and European cut-grass had the antimutagenic effect, but smartweed did not.
(4) Antimutagenic effect of each fraction fractionated with tool. wt. The extracts were fractionated into two fractions, that is, mol. wt. above and below 300 000 by ultrafiltration. The antimutagenic effect of each fraction on benzo[a]pyrene was investigated. The results are shown in Table 3. In the case of curled pondweed, the fraction
K a d a (1985) classified antimutagens into two kinds, desmutagens and bioantimutagens. DesTABLE 4 DESMUTAGENIC EFFECT OF WATER EXTRACTS F R O M PLANTS ON T H E M U T A G E N I C I T Y O F BENZO[ a ]PYRENE A m o u n t of extract (rag/plate) Spontaneous mutation Benzol a ]pyrene only Mutagen treated with water Mutagen treated with curled pondweed Mutagen treated with European cut-grass Mutagen treated with smartweed
Revertants per 10 6 survivors 9.5 37.8
0
39.0
3
20.5
3
37.3
6
19.0
TA100 ( + $ 9 mix) and 10 ~ g / p l a t e of benzo{a]pyrene were used.
216
mutagens cause chemical or biochemical modifications of mutagens outside cells, while bioantimutagens interfere with cellular functions which produce genetically stable informative genes from primary damage to DNA. The desmutagen test of 3 plants was performed and the results are shown in Table 4. These results show that the active factors of curled pondweed and smartweed may act as desmutagens. However, the active factor of European cut-grass may not act as a desmutagen and there is a possibility that the factor may be a bioantimutagen or an inhibitor of $9 mix. We have already reported that the active substance of grass-wrack pondweed acted as a desmutagen rather than a bioantimutagen (Sato et al., 1984).
on 2-nitrofluorene. Morita et al. (1984) reported that a desmutagenic factor isolated from burdock was resistant to heat and proteolytic enzyme, and had a mol. wt. above 300000. Morita et al. (1985) showed that this desmutagen might be a lignin-like compound having 10% sugar. These properties resemble those of grass-wrack pondweed and curled pondweed. The authors are investigating the chemical properties of these substances.
Discussion
Kada, T. (1973) Escherichia coli mutagenicity of furylfuramide, Jpn. J. Genet., 48, 301-305. Kada, T., T. Inoue, T. Ohta and Y. Shirasu (1985) Antimutagens and their modes of action, in: D.M. Shankel, P.E. Hartman, T. Kada and A. Hollaender (Eds.), Antimutagenesis and Anticarcinogenesis Mechanisms, Basic Life Sciences, Vol. 39, Plenum, New York, pp. 181-196. Morita, K., and T. Kada (1985) Studies on natural desmutagens: Especially, a desmutagen from burdock (Alctium lappa Linne), in: D.M. Shankel, P.E. Hartman, T. Kada and A. Hollaender (Eds.), Antimutagenesis and Anticarcinogenesis Mechanisms, Basic Life Sciences, Vol. 39, Plenum, New York, p. 569. Morita, K., T. Kada and M. Namiki (1984) A desmutagenic factor isolated from burdock, Mutation Res., 129, 25-31. Sato, T., Y. Suzuki, Y. Ose and T. Ishikawa (1984) Desmutagenic substance in water extract of grass-wrack pondweed, Mutation Res., 129, 33-38.
In previous reports, the authors reported a desmutagenic substance in grass-wrack pondweed (Sato et al., 1984). Comparisons of the antimutagenic properties of grass-wrack pondweed with those of curled pondweed, show that both properties are the same. The two plants belong to the same genus Potamogeton and a similar desmutagen may exist in these plants. While, the properties of European cut-grass and smartweed differed from Potamogeton. European cut-grass did not show the desmutagenic effect and the active substance was contained in fractions with mol. wt. both above and below 300000. The extract of smartweed did not show the antimutagenic effect
Acknowledgements The authors wish to thank our graduate student Koji Iwai for his contribution to this work.
References
211
Elsevier MTR 04353
Antimutagenic factors in aquatic plants Takako Fujimoto, Youki Ose, Takahiko Sato, Hiroaki Matsuda, Hisamitsu Nagase and Hideaki Kito
Gifu PharmaceuticalUniversity,5-6-1Mitahora-higashi, GifuCity, Gifu(Japan) (Received 31 July 1986) (Revision received 24 December 1986) (Accepted 6 January 1987)
Keywords: Aquatic plants; Antimutagenic factors; Curled pondweed; European cut-grass; Smartweed. Summary Antimutagenic activity was screened in water extracts from 13 kinds of aquatic plant and it was found that curled pondweed (Potamogeton crispus L.), European cut-grass (Leersia japonica Makino) and smartweed (Polygonum hydropiper L.) had large antimutagenic effects. Antimutagenic factors in these extracts were heat-resistant. The factors in curled pondweed and European cut-grass reduced the mutagenicities of benzo[a]pyrene and 2-nitrofluorene, but did not reduce the mutagenicity of AF-2. The factor in smartweed reduced the mutagenicity of benzo[a]pyrene, but did not reduce the mutagenicity of 2-nitrofluorene and AF-2. The factor in curled pondweed had molecular weight (mol. wt.) above 300000. The factors in European cut-grass and smartweed existed in both fractions with mol. wt. above and below 300 000. The active factors in curled pondweed and smartweed may act as the desmutagen, but that in European cut-grass does not act as a desmutagen.
The authors reported that a kind of aquatic plant, grass-wrack pondweed (Potamogeton oxyphyllus Miq.) had a desmutagenic effect and the factor was heat-resistant and had a mol. wt. above 300000 (Sato et al., 1984). In this paper, the authors collected 13 aquatic plants from some tributaries of Nagara River in Gifu Prefecture and the antimutagenic effect on benzo[a]pyrene was screened and it was found that some plants had the effect. The nature of these antimutagenic factors was investigated.
Correspondence: Dr. Takahiko Sato, Associate Professor, Department of Environmental Hygiene, Gifu Pharmaceutical University, 5-6-1 Mitahora-higashi, Gifu 502 (Japan).
Materials and methods
(1) Sampling of plants Aquatic plants were collected from some tributaries of Nagara River, such as Toba, Fukutomi and Arata Rivers in Gifu Prefecture.
(2) Preparation of water extracts Plants were washed with tap and distilled waters, minced with scissors, ground with mortar and pestle, and homogenized by a mixer with distilled water. Homogenates were centrifuged at 3500 g for 20 min and supernatants were filtered through a gauze. The filtrates were concentrated by a rotary evaporator and freeze-dried. After
0027-5107/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
212
being dissolved in proper distilled water, they were used for the experiments. For sterilization, centrifugation (1100 g, 30 min) was used at first, but later an autoclave was used, because it was known that these antimutagenic factors were heat-resistant.
(3) Test of antimutagenic effect on mutagens The antimutagenic effects of these extracts on mutagens were investigated with Salmonella typhimurium TA100 and TA98 as described by Sato et al. (1984). If metabolic activation was needed, $9 mix was added. $9 fraction was prepared from male Sprague-Dawley rats treated
with PCB (Kanechlor-400). After dilution with M / 1 5 phosphate buffer (pH 7.0), the number of surviving cells was determined by B-2 medium (Kada, 1973).
(4) Heat effect on the antimutagenic factors After sterilization by centrifugation, half of the supernatant was autoclaved at 121°C for 20 min. The antimutagenic effect on benzo[a]pyrene was investigated with the heat-treated and untreated supernatants. TA100 and 10 # g / p l a t e of benzo[a]pyrene were used and $9 mix was added. The amounts of extracts were 3 and 6 mg/plate.
TABLE 1 A N T I M U T A G E N I C E F F E C T S OF A Q U A T I C P L A N T E X T R A C T S ON B E N Z O [ a ] P Y R E N E Common name
Scientific n a m e
Smartweed
Polygonum hydropiper L.
A m o u n t of extract (mg)
Antimutagenic activity
8
-0.2 9.2
4
Curled pondweed
Potamogeton crispus L.
European cut-grass
Leersia japonica Makino
Eurasian water milfoil
Myriophyllum spicatum L.
8 4
5.6 48.2
8 4
23.1 61.0
8 4
28.8 52.2
Crowfoot
Ranunculus mpponicum Nakai var. submersus Hara
8 4
40.4 62.8
Ukishiba
Chamaeraphis spinescens Poir. var. depauperata Hook. fit.
8 4
50.2 32.2
Kougaimo
Vallisneria denseserrulata Makino
8 4
54.6 88.2
Small pondweed
Potamogeton pusillus L.
8 4
58.7 80.5
Eelgrass
Vallisneria natans Hara
8 4
59.7 69.2
Waterwort
Elatine triandra Schk. var. pedicellata Krylov
8 4
66.3 87.3
Knotweed
Polygonum thunbergii Sieb. et Zucc.
8 4
69.5 92.8
Florida elodea
Elodea nuttalli ( Flanch.) St. John
8 4
110.8 110.5
8 4
203.8 110.6
Water thyme
Hydrilla varticillata Casp.
TA100 ( + $9 mix) and 10 # g / p l a t e of benzo[a]pyrene were used.
213 TABLE 2
(6) Desmutagen test
HEAT EFFECT ON THE ANTIMUTAGENIC FACTORS
The extracts were dissolved to 30 m g / m l of water and sterilized. 2 or 4 ml of the solutions and 200 /~g of b e n z o [ a ] p y r e n e were mixed and incubated at 3 7 ° C for I h with slow shaking. Benzo[a]pyrene was extracted 3 times b y 5 ml of ethyl acetate, and the ethyl acetate was evaporated to dryness. The sample was redissolved in 2 ml of D M S O , and 0.1 ml of it was supplied to the A m e s test. It was determined beforehand that the antimutagenic substances of these plants were not extractable with ethyl acetate.
His + revertants per 10 6 survivors Curled European Smartweed pondweed cut-grass Spontaneous mutation Benzo[a]pyrene only
6.0 17.4
8.1 27.5
7.1 32.9
3 mg of extract/plate non-autoclaved autoclaved
10.1 9.6
17.3 18.0
14.4 13.9
6 mg of extract/plate non-autoclaved autoclaved
7.3 8.0
13.5 13.4
9.3 9.6
Results
(1) Screening of antimutagenic effect of water extracts on benzo[a]pyrene (5) Antimutagenic effect of fractions fractioned by moL wt. The extracts were fractionated into 2 fractions, that is, "mol. wt. above 300 000 and below 300 000 using ultrafiltration (Diaflo membrane, A m i c o n XM300). Each fraction was freeze-dried and used for the experiment. TA100 and 10 # g / p l a t e of b e n z o [ a ] p y r e n e were used and $9 mix was added. The a m o u n t of samples was 3 m g / p l a t e .
13 aquatic plants were investigated to find the antimutagenic effect on benzo[a]pyrene. F o r indication of antimutagenic effect, the antimutagenic activity was calculated by the following equation. Antimutagenic activity = (S
N)
-
xlO0
(P-N)
S, n u m b e r of revertants when the sample and
Curled pondweed
q 15
~
European c u t - g r a s s
D
5
Smartweed
lOO
5
25
50
~C .
>
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
n-
A m o u n t of e x t r a c t ( m g / p l a t e )
Fig. 1. Dose-response relationship between the amount of extracts and the mutagenicity of benzo[a]pyrene. TA100 (+ $9 mix) and 10/~g/plate of benzo[a]pyrene were used. Broken line indicates spontaneous revertants.
214
Smartweed Curled pondweed
40 w
100 >0
-
>
•
•
•
I European cut-grass
v
40
m
o
30 ~
[ •
v
I
30
•
•
•
20
4o
2t 10
g 2o I
I
I
I
2
4
6
8
10
•
..... ; ......
......
i .......
/
I
2 4 6 8 Amount of extract (mg/plote)
2
I
I
I
4
6
8
Fig. 2. D o s e - r e s p o n s e relationship between the a m o u n t of extracts and the mutagenicity of AF-2. TA100 ( + $9 mix) and 0.05 /~g/plate of AF-2 were used. Broken line indicates spontaneous revertants.
smartweed (Polygonum hydropiper L.) showed strong antimutagenic effect on benzo[a]pyrene. The properties of the antimutagenic factors were further investigated.
benzo[a]pyrene were added to TA100 simultaneously; N, number of revertants by spontaneous mutation; P, number of revertants by benzo[a]pyrene. The results are shown in Table 1. Curled pondweed (Potamogeton crispus L.), European cut-grass (Leersiajaponica Makino) and
(2) Effect of heating on the antimutagenic factors The heat resistance on antimutagenic factors in
Curled pondweed European cut-gross 40(: 300 O >
300
> o3
t ~"t• 10•"
S m o r t w e e d
q
3C !0
•
•
200
% 200
20
Q.
100 c. 100
10
i
i
i
I
2
4
6
8
I
I
I
i
2 4 6 8 Amount of extract (rag/plate)
......
I ......
1 ......
1 ......
1 ....
2
4
6
8
Fig. 3. D o s e - r e s p o n s e relationship between the a m o u n t of extracts and the mutagenicity of 2-nitrofluorene. TA100 ( + $9 mix) and 4 # g / p l a t e of 2-nitrofluorene were used. Broken line indicates spontaneous revertants.
215 TABLE 3 A N T I M U T A G E N I C E F F E C T OF T H E F R A C T I O N S W I T H mol. wt. ABOVE A N D B E L O W 3130000 ON P E N Z O [ a ] P Y R E N E His + revertants per 106 survivors
Spontaneous mutation Benzo[ a ]pyrene only Unfiltrated sample Fraction with mol. wt. above 300000 Fraction with mol. wt. below 3130000
Curled pondweed
European cut-grass
Smartweed
13.3 47.8 12.2 24.7 46.6
13.3 47.8 21.0 28.8 27.3
14.0 63.9 31.5 26.9 25.1
the extracts was examined and the results are shown in Table 2. To ascertain that the decrease of revertants did not depend on a decrease in the number of surviving cells, the number of surviving cells was determined and the frequency of revertants in survivors was calculated. In some cases, a little decrease of surviving cells numbers was observed, especially in the case of mutagens and no extract addition. There are no differences between His + revertants in survivors in 3 non-autoclaved samples and those in 3 autoclaved samples and it is clear that these antimutagenic factors are heat-resistant.
with tool. wt. above 300 000 decreased the mutagenicity of benzo[a]pyrene to about a half, but the fraction with mol. wt. below 300 000 had no effect and it can be considered that the tool. wt. of the antimutagenic factor in curled pondweed was above 300000. While in the case of European cut-grass and smartweed, the fractions with mol. wt. above and below 300 000 decreased the mutagenicity of benzo[a]pyrene to about a half and it can be considered that the mol. wt. of the antimutagenic factors in these plants is contained in fractions with mol. wt. both above and below 300 000.
(5) Desmutagen test (3) Antimutagenic effect of the extracts on various mutagens The authors examined the antimutagenic effect of the extracts on various mutagens, such as benzo[a]pyrene, 2-nitrofluorene and AF-2. The results are shown in Figs. 1, 2 and 3. The extracts of 3 plants had an antimutagenic effect on benzo[a]pyrene, but had not on AF-2. For 2-nitrofluorene, the effect differed between the three plants, that is, curled pondweed and European cut-grass had the antimutagenic effect, but smartweed did not.
(4) Antimutagenic effect of each fraction fractionated with tool. wt. The extracts were fractionated into two fractions, that is, mol. wt. above and below 300 000 by ultrafiltration. The antimutagenic effect of each fraction on benzo[a]pyrene was investigated. The results are shown in Table 3. In the case of curled pondweed, the fraction
K a d a (1985) classified antimutagens into two kinds, desmutagens and bioantimutagens. DesTABLE 4 DESMUTAGENIC EFFECT OF WATER EXTRACTS F R O M PLANTS ON T H E M U T A G E N I C I T Y O F BENZO[ a ]PYRENE A m o u n t of extract (rag/plate) Spontaneous mutation Benzol a ]pyrene only Mutagen treated with water Mutagen treated with curled pondweed Mutagen treated with European cut-grass Mutagen treated with smartweed
Revertants per 10 6 survivors 9.5 37.8
0
39.0
3
20.5
3
37.3
6
19.0
TA100 ( + $ 9 mix) and 10 ~ g / p l a t e of benzo{a]pyrene were used.
216
mutagens cause chemical or biochemical modifications of mutagens outside cells, while bioantimutagens interfere with cellular functions which produce genetically stable informative genes from primary damage to DNA. The desmutagen test of 3 plants was performed and the results are shown in Table 4. These results show that the active factors of curled pondweed and smartweed may act as desmutagens. However, the active factor of European cut-grass may not act as a desmutagen and there is a possibility that the factor may be a bioantimutagen or an inhibitor of $9 mix. We have already reported that the active substance of grass-wrack pondweed acted as a desmutagen rather than a bioantimutagen (Sato et al., 1984).
on 2-nitrofluorene. Morita et al. (1984) reported that a desmutagenic factor isolated from burdock was resistant to heat and proteolytic enzyme, and had a mol. wt. above 300000. Morita et al. (1985) showed that this desmutagen might be a lignin-like compound having 10% sugar. These properties resemble those of grass-wrack pondweed and curled pondweed. The authors are investigating the chemical properties of these substances.
Discussion
Kada, T. (1973) Escherichia coli mutagenicity of furylfuramide, Jpn. J. Genet., 48, 301-305. Kada, T., T. Inoue, T. Ohta and Y. Shirasu (1985) Antimutagens and their modes of action, in: D.M. Shankel, P.E. Hartman, T. Kada and A. Hollaender (Eds.), Antimutagenesis and Anticarcinogenesis Mechanisms, Basic Life Sciences, Vol. 39, Plenum, New York, pp. 181-196. Morita, K., and T. Kada (1985) Studies on natural desmutagens: Especially, a desmutagen from burdock (Alctium lappa Linne), in: D.M. Shankel, P.E. Hartman, T. Kada and A. Hollaender (Eds.), Antimutagenesis and Anticarcinogenesis Mechanisms, Basic Life Sciences, Vol. 39, Plenum, New York, p. 569. Morita, K., T. Kada and M. Namiki (1984) A desmutagenic factor isolated from burdock, Mutation Res., 129, 25-31. Sato, T., Y. Suzuki, Y. Ose and T. Ishikawa (1984) Desmutagenic substance in water extract of grass-wrack pondweed, Mutation Res., 129, 33-38.
In previous reports, the authors reported a desmutagenic substance in grass-wrack pondweed (Sato et al., 1984). Comparisons of the antimutagenic properties of grass-wrack pondweed with those of curled pondweed, show that both properties are the same. The two plants belong to the same genus Potamogeton and a similar desmutagen may exist in these plants. While, the properties of European cut-grass and smartweed differed from Potamogeton. European cut-grass did not show the desmutagenic effect and the active substance was contained in fractions with mol. wt. both above and below 300000. The extract of smartweed did not show the antimutagenic effect
Acknowledgements The authors wish to thank our graduate student Koji Iwai for his contribution to this work.
References