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Isolation of a mutagenic fraction from bracken (Pteridium aquilinum)
377
Mutation Research, 79 (1980) 377--380 ©Elsevier/North-Holland Biomedical Press
Short Communication ISOLATION OF A MUTAGENIC FRACTION FROM BRACKEN
(Pteridium aquilinum ) J.C.M. van der HOEVEN and F.E. van LEEUWEN
Department of Toxicology, Agricultural University, De Dreijen 12, Wageningen (The Netherlands) (Received 14 February 1980) (Revision received 24 June 1980) (Accepted 7 July 1980)
Bracken fern (Pteridium aquilinum) is a known carcinogenic plant [3--6,11, 13]. Although some mutagenic compounds occurring in bracken have been isolated, the substance(s) considered to be responsible for the carcinogenic activity of bracken is (are) still u n k n o w n [2,5--7,10,12,14,15]. Up to n o w no strong mutagenic activity of bracken fractions in vitro has been reported in the Salmonella/microsome assay and in the sister-chromatid exchange (SCE) test. This paper describes the isolation, from bracken, of a fraction that is strongly positive in the Salmonella/microsome test and in the SCE test. Fully developed fronds of bracken were collected in September 1978 and in June 1979. They were freeze-dried directly after collection. Freeze-dried bracken was extracted by the scheme described in Fig. 1. We developed this extraction scheme for detection of natural mutagens occurring in food plants. Each numbered fraction (Fig. 1) was tested for mutagenicity in the Salmonella/microsome assay [1], with strains TA100 and TA98. The only positive fraction (No. 3) was that prepared from the methanol-extract residue by solution in water and extraction of the water phase at pH 10 with ethyl acetate. This may represent the ac£ivation of mutagenic material o r the release of mutagenic material as a free form under the alkaline condition. Fraction No. 3 was prepared 8 times (with bracken collected in 1978). Each time, this fraction was tested twice for mutagenicity. To each'plate an amount of compounds derived from 120 mg freeze-dried bracken was added (Fig. 1). The mean numbers of revertants for T A 1 0 0 and TA98 (without addition of liver homogenate) were 655 (s.d. 60) and 130 (s.d. 18) resp. Spontaneous numbers, which ranged from 80 to 120 for T A 1 0 0 and from 15 to 30 for TA98, have been subtracted from the observed numbers. These data indicate that the mutagenic activity of the fraction is stronger for T A 1 0 0 than for TA98. The dose--response relationships for the 2 strains are illustrated in Fig. 2. Application of liver homogenate prepared from rats pretreated with Aroclor
378
12 g freeze-dried bracken
b
extracted with 400 ml light petroleum for 2 h
I
i
I
bracken residue extracted 3 times with 400 ml methanol for 3 h
light petroleum evaporated (1)
I methanol
extract
bracken residue !xtracted with 400 ml water for 3 h
divided into
3 equal parts
I
water evaporated (5) 1/3
J
J__
methanol evaporated (2)
113
~] / 3 1 methanol
evaporated
and
r e s i d u e d i s s o l v e d in w a t e r ; solution extracted with ethyl acetate at pH 10; ethyl acetate evaporated (3)
methanol evaporated, residue dissolved in water; solution extracted with ethyl acetate at pH 2; ethyl acetate evaporated (4)
Fig. 1. E x t r a c t i o n s c h e m e . E a c h n u m b e r e d f r a c t i o n was dissolved in DMSO a n d t e s t e d f o r m u t a g e r d c i t y in the S a l m o n e l l a / m i c r o s o m e assay. F r a c t i o n Nos. 1 a n d 5 were dissolved in 10 m l D M S O , w h e r e a s Nos. 2, 3 a n d 4 were dissolved in 3.3 m l DMSO. Because 0.1 m l DMSO was a d d e d to t h e plates, e v e r y p l a t e was e x p o s e d to a n a m o u n t of c o m p o u n d s r e p r e s e n t i n g 1 2 0 m g f r e e z e - d r i e d b r a c k e n . E x t r a c t i o n s w e r e c a r r i e d o u t in a S o x h l e t a p p a r a t u s . E v a p o r a t i o n s w e r e c a r r i e d o u t in a r o t a r y e v a p o r a t o r .
200 j
50
A
7J" A
go
ab
~2o
D
SCE
ler chromosome
20 600-
×
jx
15
jx
o 1.0 o 200 -
o
0.5-
4'o
8'0
1~o
dose (llg freeze-dried bracken per plote)
dese(mg freeze- dried brocken/ml)
Fig. 2.
D o s e - - r e s p o n s e r e l a t i o n s h i p s for t h e m u t a g e n i c i t y o f f r a c t i o n No. 3 ( b r a c k e n c o l l e c t e d in 1 9 7 9 ) ; ~, T A 9 8 - - $ 9 ; A T A 9 8 + $ 9 ; o, T A 1 0 0 - - S 9 ; e, T A 1 0 0 + S 9 . T h e n u m b e r s o f r e v e r t a n t s are averages f r o m 2 Expts.
Fig. 3. D o s e - - r e s p o n s e r e l a t i o n s h i p for t h e i n d u c t i o n of SCEs b y f r a c t i o n No. 3 ( b r a c k e n c o l l e c t e d in Sept e m b e r 1 9 7 8 ) . SCEs w e r e p r e p a r e d b y a slight m o d i f i c a t i o n o f t h e m e t h o d o f G o t o et al. [ 8 ] . 50 0 0 0 V 7 9 Chinese h a m s t e r cells w e r e s e e d e d o n s i d e s ( 2 0 c m 2) w h i c h w e r e p l a c e d in 1 0 - c m p e t r i dishes c o n t a i n i n g 10 m l Eagle's basal m e d i u m s u p p l e m e n t e d w i t h 10% n e w b o r n calf s e r u m . 24 h later, B r d U a n d b r a c k e n e x t r a c t w e r e a d d e d . Colchicine w a s a d d e d 5 h b e f o r e swelling a n d f i x a t i o n o f t h e cells. T h e t o t a l e x p o s u r e t i m e t o B r d U a n d b r a c k e n e x t r a c t w a s 32 h. T h e test p r o c e d u r e w a s r e p e a t e d o n c e , a n d e a c h dose level was t e s t e d in d u p l i c a t e . 25 m e t a p h a s e s p e r slide w e r e s c o r e d . S t a n d a r d d e v i a t i o n s r a n g e d f r o m 0 . 1 6 SCEs p e r c h r o m o s o m e for t h e c o n t r o l t o 0 . 6 7 2 SCEs p e r c h r o m o s o m e for t h e h i g h e s t dose level.
379
TABLE
1
THE INFLUENCE OF pH ON THE MUTAGENICITY Bracken collected in 1978. pH of the water phase
2 5 b 7 8 10 10 c
Revertants
OF BRACKEN
FRACTION
No. 2
per plate a
TA98
TA100
0 8 19 30 120 70
0 60 80 200 570 440
a Spontaneous numbers are subtracted. b pH not adjusted. c P r e - i n c u b a t e d a t p H 2.
1254 according to the method of Ames et al. [1] increased the number of revertants for TA98 but decreased the number of revertants for TA100, probably by deactivation. Storage of the active fraction at 4°C for several days led to a substantial loss of mutagenic activity. The mutagenic fraction was also tested in the SCE test with V79 Chinese hamster cells (Fig. 3). There was a positive response. Fractions that were negative in the Salmonella/microsome assay were not tested in the SCE test. To investigate the influence of pH on the mutagenic activity in the Salmonella/microsome assay, fractions were prepared as follows. Fraction No. 2 (Fig. 1) was dissolved in water and extracted with ethyl acetate at various pH levels. As can be seen in Table 1, the mutagenicity of bracken was pH-dependent. Because the mutagenic principle(s) had been extracted into ethyl acetate under alkaline conditions it may be assumed that the substance(s) has (have) one or more basic groups. This has not yet been reported by other workers in this field. It is of interest to mention that, in the rat, most bracken-induced intestinal adenocarcinomas have been reported to occur in the ileal region [3,13]. Hirono et al. [9] found that the most commonest site of these tumours was the terminal 3-cm section of the ileum. This is where the highest pH is found. Our data support the hypothesis that a basic fraction is involved in the process of bracken carcinogenesis. Furthermore, these resultS may explain the observation that treatment with alkali decreases the carcinogenic properties of bracken and bracken extracts [6,9]. These results may lead to the "isolation and identification of bracken mutagens and carcinogens by using short-term mutagenicity tests. Investigations to isolate and identify the mutagenic compound(s) are in progress in our laboratory. This research was supported by a grant from the Netherlands Cancer Society (Koningin Wilhelmina Fonds).
380
References 1 A m e s , B.N., J. M c C a n n a n d E. Y a r n a s a k i , M e t h o d s f o r d e t e c t i n g c a r c i n o g e n s a n d r n u t a g e n s w i t h t h e S a l r n o n e l l a / r n a r n m a l i a n - m i c r o s o m e r n u t a g e n i c i t y test, M u t a t i o n Res., 31 ( 1 9 7 5 ) 3 4 7 - - 3 6 4 . 2 B a r b e r , G . D . , P h . D . thesis, U n i v e r s i t y o f Wales 1 9 6 9 ; c i t e d in E v a n s [ 6 ] . 3 E v a n s , I.A., a n d J. M a s o n , C a r c i n o g e n i c a c t i v i t y o f b r a c k e n , N a t u r e ( L o n d o n ) , 2 0 8 ( 1 9 6 5 ) 9 1 3 - - 9 1 4 . 4 Evans, I.A., T h e r a d i o m i r n e t i c n a t u r e o f b r a c k e n t o x i n , C a n c e r R e s . , 2 8 ( 1 9 6 8 ) 2 2 5 2 - - 2 2 6 1 . 5 E v a n s , I.A., a n d M.A. O s r n a n , C a r c i n o g e n i c i t y o f b r a c k e n a n d s h i k i m i c a c i d , N a t u r e ( L o n d o n ) , 2 5 0 (1974) 348. 6 Evans, I.A., B r a c k e n c a r c i n o g e n i c i t y , Res. V e t e r i n a r y Sci., 2 6 ( 1 9 7 9 ) 3 3 9 - - 3 4 8 . 7 F u k u o k a , M., M. K t t r o y a n a g i , K. Y o s h i h l r a , S. N a t o r i , M. N a g a o , Y. T a k a h a s h i a n d T. S u g i r n u r a , C h e m i c a l a n d t o x i c o l o g i c a l s t u d i e s o n b r a c k e n f e r n , Pteridium aquflinum var. Latiusculum, IV. Surv e y s o n b r a c k e n c o n s t i t u e n t s b y r n u t a g e n test, J. P h a r r n . D y n . , 1 ( 1 9 7 8 ) 3 2 4 - - 3 3 1 . 8 G o t o , K., T. A k e r n a t s u , H. S h i m a z u a n d T. S u g i y a r n a , S i m p l e d i f f e r e n t i a l G i e m s a s t a i n i n g o f sister chrornatids after treatment with photosensitive dyes and exposure to light and the mechanism of staining, Chromosoma, 53 (1975) 223--230. 9 H i r o n o , L., C. S h i b u y a , M. S h i r n i z u a n d K. F u s h i m i , C a r c i n o g e n i c a c t i v i t y o f p r o c e s s e d b r a c k e n u s e d as h u m a n f o o d , J. N a t l . C a n c e r Inst., 4 8 ( 1 9 7 2 ) 1 2 4 5 - - 1 2 5 0 . 1 0 H i r o n o , J., K. F u s h i m i a n d N. M a t s u b a r a , C a ~ c i n o g e n i c i t y t e s t o f s h i k i m i c a c i d in r a t s , T o x i c o l . L e t t . , (1977) 9--10. 11 P a r n u k c u , A . M . , a n d J.M. Price, I n d u c t i o n o f i n t e s t i n a l a n d u r i n a r y b l a d d e r c a n c e r in r a t s b y f e e d i n g b r a c k e n f e r n (Pteris aqulina), J. N a t l . C a n c e r Inst., 4 3 ( 1 9 6 9 ) 2 7 5 - - 2 8 1 . 1 2 P a r n u k c u , A.M., E. E z t i i r k , S. Y a l c i n e r , U. Milli a n d G . T . B r y a n , C a r c i n o g e n i c a n d m u t a g e n i c a c t i v i t i e s o f m i l k f r o m c o w s f e d b r a c k e n f e r n (Pteridium aquilinum), C a n c e r Res., 3 8 ( 1 9 7 8 ) 1 5 5 6 - - 1 5 6 0 . 1 3 Price, J . M . , a n d A.M. P a m u k c u , T h e i n d u c t i o n o f n e o p l a s m s o f t h e u r i n a r y b l a d d e r o f t h e c o w a n d t h e s m a l l i n t e s t i n e o f t h e r a t b y f e e d i n g b r a c k e n f e r n , C a n c e r Res., 2 8 ( 1 9 6 8 ) 2 2 4 7 - - 2 2 5 1 . 1 4 R o b e r t s , I.M., D.S. S h a w a n d W.C. E v a n s , A T 4 - b a c t e r i o p h a g e r e v e r s i o n t e s t f o r t h e n a t u r a l l y o c c u r r i n g m u t a g e n p r e s e n t in b r a c k e n f e r n (Pteridium aquiZinum), B i o c h e m . J., 1 2 4 ( 1 9 7 1 ) 1 3 p . 1 5 S u g i r n u r a , T., M. N a g a o , T. M a t s n s h i m a , T. Y a h a g i , Y. S e i n o , A. Shiral, M. S a w a m u r a , S. N a t o r i , K. Y o s h i h i r a , M. F u k u o k a a n d M. K t t r o y a n a g i , M u t a g e n i c i t y o f f l a v o n e d e r i v a t i v e s , P r o c . J p n . A c a d . , 53, Ser. B ( 1 9 7 7 ) 1 9 4 - - 1 9 7 .
Mutation Research, 79 (1980) 377--380 ©Elsevier/North-Holland Biomedical Press
Short Communication ISOLATION OF A MUTAGENIC FRACTION FROM BRACKEN
(Pteridium aquilinum ) J.C.M. van der HOEVEN and F.E. van LEEUWEN
Department of Toxicology, Agricultural University, De Dreijen 12, Wageningen (The Netherlands) (Received 14 February 1980) (Revision received 24 June 1980) (Accepted 7 July 1980)
Bracken fern (Pteridium aquilinum) is a known carcinogenic plant [3--6,11, 13]. Although some mutagenic compounds occurring in bracken have been isolated, the substance(s) considered to be responsible for the carcinogenic activity of bracken is (are) still u n k n o w n [2,5--7,10,12,14,15]. Up to n o w no strong mutagenic activity of bracken fractions in vitro has been reported in the Salmonella/microsome assay and in the sister-chromatid exchange (SCE) test. This paper describes the isolation, from bracken, of a fraction that is strongly positive in the Salmonella/microsome test and in the SCE test. Fully developed fronds of bracken were collected in September 1978 and in June 1979. They were freeze-dried directly after collection. Freeze-dried bracken was extracted by the scheme described in Fig. 1. We developed this extraction scheme for detection of natural mutagens occurring in food plants. Each numbered fraction (Fig. 1) was tested for mutagenicity in the Salmonella/microsome assay [1], with strains TA100 and TA98. The only positive fraction (No. 3) was that prepared from the methanol-extract residue by solution in water and extraction of the water phase at pH 10 with ethyl acetate. This may represent the ac£ivation of mutagenic material o r the release of mutagenic material as a free form under the alkaline condition. Fraction No. 3 was prepared 8 times (with bracken collected in 1978). Each time, this fraction was tested twice for mutagenicity. To each'plate an amount of compounds derived from 120 mg freeze-dried bracken was added (Fig. 1). The mean numbers of revertants for T A 1 0 0 and TA98 (without addition of liver homogenate) were 655 (s.d. 60) and 130 (s.d. 18) resp. Spontaneous numbers, which ranged from 80 to 120 for T A 1 0 0 and from 15 to 30 for TA98, have been subtracted from the observed numbers. These data indicate that the mutagenic activity of the fraction is stronger for T A 1 0 0 than for TA98. The dose--response relationships for the 2 strains are illustrated in Fig. 2. Application of liver homogenate prepared from rats pretreated with Aroclor
378
12 g freeze-dried bracken
b
extracted with 400 ml light petroleum for 2 h
I
i
I
bracken residue extracted 3 times with 400 ml methanol for 3 h
light petroleum evaporated (1)
I methanol
extract
bracken residue !xtracted with 400 ml water for 3 h
divided into
3 equal parts
I
water evaporated (5) 1/3
J
J__
methanol evaporated (2)
113
~] / 3 1 methanol
evaporated
and
r e s i d u e d i s s o l v e d in w a t e r ; solution extracted with ethyl acetate at pH 10; ethyl acetate evaporated (3)
methanol evaporated, residue dissolved in water; solution extracted with ethyl acetate at pH 2; ethyl acetate evaporated (4)
Fig. 1. E x t r a c t i o n s c h e m e . E a c h n u m b e r e d f r a c t i o n was dissolved in DMSO a n d t e s t e d f o r m u t a g e r d c i t y in the S a l m o n e l l a / m i c r o s o m e assay. F r a c t i o n Nos. 1 a n d 5 were dissolved in 10 m l D M S O , w h e r e a s Nos. 2, 3 a n d 4 were dissolved in 3.3 m l DMSO. Because 0.1 m l DMSO was a d d e d to t h e plates, e v e r y p l a t e was e x p o s e d to a n a m o u n t of c o m p o u n d s r e p r e s e n t i n g 1 2 0 m g f r e e z e - d r i e d b r a c k e n . E x t r a c t i o n s w e r e c a r r i e d o u t in a S o x h l e t a p p a r a t u s . E v a p o r a t i o n s w e r e c a r r i e d o u t in a r o t a r y e v a p o r a t o r .
200 j
50
A
7J" A
go
ab
~2o
D
SCE
ler chromosome
20 600-
×
jx
15
jx
o 1.0 o 200 -
o
0.5-
4'o
8'0
1~o
dose (llg freeze-dried bracken per plote)
dese(mg freeze- dried brocken/ml)
Fig. 2.
D o s e - - r e s p o n s e r e l a t i o n s h i p s for t h e m u t a g e n i c i t y o f f r a c t i o n No. 3 ( b r a c k e n c o l l e c t e d in 1 9 7 9 ) ; ~, T A 9 8 - - $ 9 ; A T A 9 8 + $ 9 ; o, T A 1 0 0 - - S 9 ; e, T A 1 0 0 + S 9 . T h e n u m b e r s o f r e v e r t a n t s are averages f r o m 2 Expts.
Fig. 3. D o s e - - r e s p o n s e r e l a t i o n s h i p for t h e i n d u c t i o n of SCEs b y f r a c t i o n No. 3 ( b r a c k e n c o l l e c t e d in Sept e m b e r 1 9 7 8 ) . SCEs w e r e p r e p a r e d b y a slight m o d i f i c a t i o n o f t h e m e t h o d o f G o t o et al. [ 8 ] . 50 0 0 0 V 7 9 Chinese h a m s t e r cells w e r e s e e d e d o n s i d e s ( 2 0 c m 2) w h i c h w e r e p l a c e d in 1 0 - c m p e t r i dishes c o n t a i n i n g 10 m l Eagle's basal m e d i u m s u p p l e m e n t e d w i t h 10% n e w b o r n calf s e r u m . 24 h later, B r d U a n d b r a c k e n e x t r a c t w e r e a d d e d . Colchicine w a s a d d e d 5 h b e f o r e swelling a n d f i x a t i o n o f t h e cells. T h e t o t a l e x p o s u r e t i m e t o B r d U a n d b r a c k e n e x t r a c t w a s 32 h. T h e test p r o c e d u r e w a s r e p e a t e d o n c e , a n d e a c h dose level was t e s t e d in d u p l i c a t e . 25 m e t a p h a s e s p e r slide w e r e s c o r e d . S t a n d a r d d e v i a t i o n s r a n g e d f r o m 0 . 1 6 SCEs p e r c h r o m o s o m e for t h e c o n t r o l t o 0 . 6 7 2 SCEs p e r c h r o m o s o m e for t h e h i g h e s t dose level.
379
TABLE
1
THE INFLUENCE OF pH ON THE MUTAGENICITY Bracken collected in 1978. pH of the water phase
2 5 b 7 8 10 10 c
Revertants
OF BRACKEN
FRACTION
No. 2
per plate a
TA98
TA100
0 8 19 30 120 70
0 60 80 200 570 440
a Spontaneous numbers are subtracted. b pH not adjusted. c P r e - i n c u b a t e d a t p H 2.
1254 according to the method of Ames et al. [1] increased the number of revertants for TA98 but decreased the number of revertants for TA100, probably by deactivation. Storage of the active fraction at 4°C for several days led to a substantial loss of mutagenic activity. The mutagenic fraction was also tested in the SCE test with V79 Chinese hamster cells (Fig. 3). There was a positive response. Fractions that were negative in the Salmonella/microsome assay were not tested in the SCE test. To investigate the influence of pH on the mutagenic activity in the Salmonella/microsome assay, fractions were prepared as follows. Fraction No. 2 (Fig. 1) was dissolved in water and extracted with ethyl acetate at various pH levels. As can be seen in Table 1, the mutagenicity of bracken was pH-dependent. Because the mutagenic principle(s) had been extracted into ethyl acetate under alkaline conditions it may be assumed that the substance(s) has (have) one or more basic groups. This has not yet been reported by other workers in this field. It is of interest to mention that, in the rat, most bracken-induced intestinal adenocarcinomas have been reported to occur in the ileal region [3,13]. Hirono et al. [9] found that the most commonest site of these tumours was the terminal 3-cm section of the ileum. This is where the highest pH is found. Our data support the hypothesis that a basic fraction is involved in the process of bracken carcinogenesis. Furthermore, these resultS may explain the observation that treatment with alkali decreases the carcinogenic properties of bracken and bracken extracts [6,9]. These results may lead to the "isolation and identification of bracken mutagens and carcinogens by using short-term mutagenicity tests. Investigations to isolate and identify the mutagenic compound(s) are in progress in our laboratory. This research was supported by a grant from the Netherlands Cancer Society (Koningin Wilhelmina Fonds).
380
References 1 A m e s , B.N., J. M c C a n n a n d E. Y a r n a s a k i , M e t h o d s f o r d e t e c t i n g c a r c i n o g e n s a n d r n u t a g e n s w i t h t h e S a l r n o n e l l a / r n a r n m a l i a n - m i c r o s o m e r n u t a g e n i c i t y test, M u t a t i o n Res., 31 ( 1 9 7 5 ) 3 4 7 - - 3 6 4 . 2 B a r b e r , G . D . , P h . D . thesis, U n i v e r s i t y o f Wales 1 9 6 9 ; c i t e d in E v a n s [ 6 ] . 3 E v a n s , I.A., a n d J. M a s o n , C a r c i n o g e n i c a c t i v i t y o f b r a c k e n , N a t u r e ( L o n d o n ) , 2 0 8 ( 1 9 6 5 ) 9 1 3 - - 9 1 4 . 4 Evans, I.A., T h e r a d i o m i r n e t i c n a t u r e o f b r a c k e n t o x i n , C a n c e r R e s . , 2 8 ( 1 9 6 8 ) 2 2 5 2 - - 2 2 6 1 . 5 E v a n s , I.A., a n d M.A. O s r n a n , C a r c i n o g e n i c i t y o f b r a c k e n a n d s h i k i m i c a c i d , N a t u r e ( L o n d o n ) , 2 5 0 (1974) 348. 6 Evans, I.A., B r a c k e n c a r c i n o g e n i c i t y , Res. V e t e r i n a r y Sci., 2 6 ( 1 9 7 9 ) 3 3 9 - - 3 4 8 . 7 F u k u o k a , M., M. K t t r o y a n a g i , K. Y o s h i h l r a , S. N a t o r i , M. N a g a o , Y. T a k a h a s h i a n d T. S u g i r n u r a , C h e m i c a l a n d t o x i c o l o g i c a l s t u d i e s o n b r a c k e n f e r n , Pteridium aquflinum var. Latiusculum, IV. Surv e y s o n b r a c k e n c o n s t i t u e n t s b y r n u t a g e n test, J. P h a r r n . D y n . , 1 ( 1 9 7 8 ) 3 2 4 - - 3 3 1 . 8 G o t o , K., T. A k e r n a t s u , H. S h i m a z u a n d T. S u g i y a r n a , S i m p l e d i f f e r e n t i a l G i e m s a s t a i n i n g o f sister chrornatids after treatment with photosensitive dyes and exposure to light and the mechanism of staining, Chromosoma, 53 (1975) 223--230. 9 H i r o n o , L., C. S h i b u y a , M. S h i r n i z u a n d K. F u s h i m i , C a r c i n o g e n i c a c t i v i t y o f p r o c e s s e d b r a c k e n u s e d as h u m a n f o o d , J. N a t l . C a n c e r Inst., 4 8 ( 1 9 7 2 ) 1 2 4 5 - - 1 2 5 0 . 1 0 H i r o n o , J., K. F u s h i m i a n d N. M a t s u b a r a , C a ~ c i n o g e n i c i t y t e s t o f s h i k i m i c a c i d in r a t s , T o x i c o l . L e t t . , (1977) 9--10. 11 P a r n u k c u , A . M . , a n d J.M. Price, I n d u c t i o n o f i n t e s t i n a l a n d u r i n a r y b l a d d e r c a n c e r in r a t s b y f e e d i n g b r a c k e n f e r n (Pteris aqulina), J. N a t l . C a n c e r Inst., 4 3 ( 1 9 6 9 ) 2 7 5 - - 2 8 1 . 1 2 P a r n u k c u , A.M., E. E z t i i r k , S. Y a l c i n e r , U. Milli a n d G . T . B r y a n , C a r c i n o g e n i c a n d m u t a g e n i c a c t i v i t i e s o f m i l k f r o m c o w s f e d b r a c k e n f e r n (Pteridium aquilinum), C a n c e r Res., 3 8 ( 1 9 7 8 ) 1 5 5 6 - - 1 5 6 0 . 1 3 Price, J . M . , a n d A.M. P a m u k c u , T h e i n d u c t i o n o f n e o p l a s m s o f t h e u r i n a r y b l a d d e r o f t h e c o w a n d t h e s m a l l i n t e s t i n e o f t h e r a t b y f e e d i n g b r a c k e n f e r n , C a n c e r Res., 2 8 ( 1 9 6 8 ) 2 2 4 7 - - 2 2 5 1 . 1 4 R o b e r t s , I.M., D.S. S h a w a n d W.C. E v a n s , A T 4 - b a c t e r i o p h a g e r e v e r s i o n t e s t f o r t h e n a t u r a l l y o c c u r r i n g m u t a g e n p r e s e n t in b r a c k e n f e r n (Pteridium aquiZinum), B i o c h e m . J., 1 2 4 ( 1 9 7 1 ) 1 3 p . 1 5 S u g i r n u r a , T., M. N a g a o , T. M a t s n s h i m a , T. Y a h a g i , Y. S e i n o , A. Shiral, M. S a w a m u r a , S. N a t o r i , K. Y o s h i h i r a , M. F u k u o k a a n d M. K t t r o y a n a g i , M u t a g e n i c i t y o f f l a v o n e d e r i v a t i v e s , P r o c . J p n . A c a d . , 53, Ser. B ( 1 9 7 7 ) 1 9 4 - - 1 9 7 .