191
Mutation Research, 48 (1977) 191--194 © Elsevier/North-Holland Biomedical Press
MUTAGENIC E F F E C T OF A R G E M O N E OIL IN OR Y Z A SA T I V A L.
T.P. REDDY and K. VAIDYANATH Department of Genetics, Osmania University, Hyderabad, 500 007 A.P. (India)
(Received July 27th, 1976) (Revision received October 6th, 1976) (Accepted October 20th, 1976)
Summary In view of the large scale adulteration of edible oils with Argemone oil, a study was undertaken to test its mutagenicity on rice seed. In treatment with screw-pressed oil a marked reduction in seed germination and high chlorophyll mutation and m u t a n t frequencies were observed. In sequential treatments be° tween gamma rays and Argemone oil both mutation and mutant frequencies were significantly higher than that of individual treatments. This study clearly indicates that Argemone oil is a p o t e n t mutagen with serious implications f o r the genetic hygiene of human beings.
Introduction The seeds of Argemone mexicana L. yield a nauseous, bitter and n o n ~ d i b l e oil which is known to cause vomitting and purging at higher doses. The oil is often used as an adulterant of mustard and other vegetable oils that are used as cooking media [1]. It has been well established that edible oils mixed with the Argemone oil cause epidemic dropsy [ 5]. In recent investigations, Ranadive et al. [8] have proved the co-carcinogenetic effects of Argemone oil in mustard oil samples from the market. Subramanyam et al. [12] studied the cytological effects of Argemone oil on the mitotic cells of Allium cepa, the screw-pressed oil was more effective in inducing chromosomal aberrations than was the solvent~extracted oil. In view of the absence of information on the possible mutagenic effect of Argemone oil, a study was undertaken to see the effect of screwpressed oil on rice seed. Materials and methods A set of 200 dehusked rice seeds (cultivar IET-1991) was pre-soaked in distilled water for 2 h before treatment with Argemone oil for 16 h. A second set
192 of seeds, immersed in distilled water for 18 h, served as control. These treatments were repeated four times under identical conditions on different days. In a second experiment, two sets of dehusked seeds were irradiated with gamma rays at 20 kR; one set was soaked in H20 for 18 h, the second set was soaked in HsO for 2 h and then immersed in Argemone oil for 16 h. In yet another experiment two sets of dehusked seeds were immersed in HsO (18 h) or oil (2 h H20 + 16 h oil) before irradiation with 20 kR gamma rays. After completion of the treatment, the excess oil was removed from seeds which were then washed thoroughly in water for about 20 min. After ten days in petri dishes, germination counts were taken, and seedlings were transplanted into pots. The selfed panicles, at the rate of 250 to 300 per treatment, were collected from M1 plants. The Ms generation was raised in panicle progenies, and data on chlorophyll mutation and mutant frequencies were collected based on M~ segregating panicles and Ms mutant seedlings, respectively. The differences in the mutation and mutant frequencies were tested by the standard normal deviate (Z) test. Results and discussion As shown in Table I, a marked reduction in seed germination was induced by Argemone oil in comparison with seeds soaked in H20 (Expt. 1). A similar trend was observed for reduction in seed germination in combination treatments between gamma rays and Argemone oil compared with irradiated seeds soaked in HsO (Table I). The results obtained in the replicated Expt. 1 indicate that screw-pressed oil is a fairly potent mutagen; the mean chlorophyll mutation and mutant frequencies were 13.52 and 1.86%, respectively.
TABLE
I
FREQUENCY OF CHLOROPHYLL AND SEQUENTIAL TREATMENTS
Seed Get mio nation (%)
Treatment
Expt. 1 H20 18 h
MUTATIONS INDUCED BY ARGEMONE WITH GAMMA RAYS IN RICE
Total number of M 1 panicles analysed
Mutation frequency % -+ S.D.
--
OIL IN INDIVIDUAL
Total number of M2 seedlings scored
Mutant frequency % ± S.D.
94.8
1248
46,011
--
72.4
1158
1 3 . 5 2 +- 1 . 9 2
39,930
1 . 8 6 -+ 0 . 1 4 7
Expt. 2 20 kR + H20 18 h
82.5
263
1 0 . 5 9 -+ 1 . 6 2
8721
1 . 4 2 +- 0 . 1 0 2
20kR+ H20 + oil 1 6 h
H20
2 h + oil 1 6 h
2h 53.0
252
2 2 . 7 8 -+ 2 . 4 3 a
6192
3 . 7 3 -+ 0 , 1 7 2 a
Expt. 3 H20 18 h + 20 kR
64.5
261
1 8 . 4 6 -+ 2 . 2 1
7563
2.38 + 0.163
H20 2h+ oi116 h + 20 kR
39.5
250
2 9 . 1 7 -+ 2 . 9 3 b
5816
4 . 0 9 -+ 0 . 1 6 9 a
a Significant at P = 0 . 0 0 1 . b Significant at P = 0 . 0 1 .
193 In Expt. 2, significant increases in mutation and mutant frequencies were observed when irradiated seeds were immersed in oil as compared with watersoaked seeds. Similarly, the set of seeds pre-soaked in oil before irradiation showed significantly higher mutation and mutant frequencies compared with water pre-soaked seeds. In sequential treatments with oil and gamma rays both mutation and mutant frequencies were almost additive (Table I). The oil extracted from mustard seeds, containing a sufficient quantity of Argemone seeds, is known to cause erythema, epidemic dropsy, gastro-intestinal disturbances, hepatitis, oedema, cardiac failure, abortion, glaucoma and even cancer [2--9,11]. The toxic effects of oil were attributed to an alkaloid, namely sanguinarine, present in it [10]. Recent investigations have shown that a fatty acid factor is essential to potentiate the toxic effects of sanguinarine [6,7,9,11]. In L-cells, chromosomal aberrations were induced by treatment with sanguinarine [13]. Treatment of onion root tips with screw-pressed oil induced extensive chromosomal damage [12]. The observations made in this study prove conclusively that screw-pressed Argemone oil is a potent mutagen. The induction of mutations by Argemone oil in multicellular organisms has serious implications for the genetic hygiene of human beings, as it is being used for large scale adulteration of vegetable oils used as cooking media in India and elsewhere. Acknowledgements The authors gratefully acknowledge the encouragement given by Dr. O.S. Reddi and Dr. G.M. Reddy of the Department of Genetics, Osmania University. References 1 B h a t n a g a r , S.S., R . N . C h o p r a , B. P r a s a d , J . C . G o s h , M. Q u e r e s h i , M.N. S a h a , L. S r i r a m a n d B . L . M a n j u n a t h , T h e W e a l t h o f I n d i a , Vol. A - - B . , ( A d i c t i o n a r y of I n d i a n r a w m a t e r i a l s a n d i n d u s t r i a l p r o d ucts) published by the Council of Scientific and Industrial Research, 1948. 2 Lal, S.B., E p i d e m i c d r o p s y in B i h a r , I n d . Med. G a z . , 8 6 ( 1 9 5 1 ) 6 4 - - 7 1 . 3 Lal, R . B . a n d R . B . D a s g u p t a , I n v e s t i g a t i o n s i n t o the e p i d e m i o l o g y o f e p i d e m i c d r o p s y . X. A n o t e o n a n o u t b r e a k o f e p i d e m i c d r o p s y a s s o c i a t e d w i t h t h e use of m u s t a r d oil pressed f r o m seeds, a d u l t e r a t e d w i t h s e e d s o f A r g e m o n e m e x i c a n a , I n d . J o u r . Med. Res., 2 9 ( 1 9 4 1 ) 1 5 7 - - 1 6 5 . 4 L a l , R . B . a n d S.C. R o y , I n v e s t i g a t i o n i n t o the e p i d e m i o l o g y o f e p i d e m i c d r o p s y . I. I n t r o d u c t o r y n o t e s a n d h i s t o r i c a l s u r v e y , I n d . J o u r . Med. Res., 2 5 ( 1 9 3 7 ) 1 6 3 - - 1 7 6 . 5 P a t w a r d h a n , V . N . , in " N u t r i t i o n in I n d i a " p u b l i s h e d b y Dr. J . C . Patel, f o r I n d i a n J o u r n a l o f M e d i c a l Sciences (1952). 6 R a m a s h a s t r i , B . V . a n d S. B a b u , A r g e m o n e t o x i c i t y , A n n u a l r e p o r t , N a t i o n a l I n s t i t u t e o f N u t r i t i o n , H y d e r a b a d , 1 9 6 9 , p. 34. 7 R a m a s h a s t r i , B . V . a n d S. B a b u , A r g e m o n e t o x i c i t y , A n n u a l r e p o r t , N a t i o n a l I n s t i t u t e o f N u t r i t i o n , H y d e r a b a d , 1 9 7 0 , p. 31. 8 R a n a d i v e , K . J . , S . V . C o t h o s k a r a n d B.U. T e z a b w a l a , T e s t i n g c a r c i n o g e n i c i t y o f c o n t a m i n a n t s in edible oils. II. A r g e m o n e off in m u s t a r d oil, I n d . J o u r . Med. Res., 61 ( 1 9 7 3 ) 4 2 8 - - 4 3 4 . 9 R u k m i n i , C., S a n g u i n a r i n e p o t e n t i a t i n g f a c t o r in A r g e m o n e oil, I n d . J o u r . Med. Res., 59 ( 1 9 7 1 ) 1676--1680. 1 0 S a r k a r , S.N., I s o l a t i o n f r o m A r g e m o n e oil o f d i h y d r o s a n g u i n a r i n e a n d s a n g u i n a r i n e : t o x i c i t y o f sanguinarine, Nature (London), 162 (1948) 265--266. 11 S h e n o l i k a z , L.S., A r g e m o n e t o x i c i t y , Annuv.l r e p o r t , N a t i o n a l I n s t i t u t e o f N u t r i t i o n , H y d e r a b a d , 1 9 7 1 , p. 49. 1 2 S u b r a m a n y a m , S.S., P. V e n k a t R e d d y , G . P r e m v e e r R e d d y a n d D w a r a k a n a t h , K . M u r t h y , C y t o l o g i e a l e f f e c t s o f A r g e m o n e oil o n t h e m i t o t i c cells o f A l l i u m cepa, P r o c . I n d . A c a d . Sci., 7 9 ( 1 9 7 4 ) 2 1 6 - 226. 1 3 Svejda, J . J . , S . R . D v e r a k a n d R . A d a m e k , C h a n g e s in L-cells after t r e a t m e n t w i t h s o m e ( i s o c h i n o l i n e ) a l k a l o i d s , Scr. M e d . ( B r n o ) , 4 2 ( 1 9 6 9 ) 2 9 1 - - 2 9 5 .
Mutation Research, 48 (1977) 191--194 © Elsevier/North-Holland Biomedical Press
MUTAGENIC E F F E C T OF A R G E M O N E OIL IN OR Y Z A SA T I V A L.
T.P. REDDY and K. VAIDYANATH Department of Genetics, Osmania University, Hyderabad, 500 007 A.P. (India)
(Received July 27th, 1976) (Revision received October 6th, 1976) (Accepted October 20th, 1976)
Summary In view of the large scale adulteration of edible oils with Argemone oil, a study was undertaken to test its mutagenicity on rice seed. In treatment with screw-pressed oil a marked reduction in seed germination and high chlorophyll mutation and m u t a n t frequencies were observed. In sequential treatments be° tween gamma rays and Argemone oil both mutation and mutant frequencies were significantly higher than that of individual treatments. This study clearly indicates that Argemone oil is a p o t e n t mutagen with serious implications f o r the genetic hygiene of human beings.
Introduction The seeds of Argemone mexicana L. yield a nauseous, bitter and n o n ~ d i b l e oil which is known to cause vomitting and purging at higher doses. The oil is often used as an adulterant of mustard and other vegetable oils that are used as cooking media [1]. It has been well established that edible oils mixed with the Argemone oil cause epidemic dropsy [ 5]. In recent investigations, Ranadive et al. [8] have proved the co-carcinogenetic effects of Argemone oil in mustard oil samples from the market. Subramanyam et al. [12] studied the cytological effects of Argemone oil on the mitotic cells of Allium cepa, the screw-pressed oil was more effective in inducing chromosomal aberrations than was the solvent~extracted oil. In view of the absence of information on the possible mutagenic effect of Argemone oil, a study was undertaken to see the effect of screwpressed oil on rice seed. Materials and methods A set of 200 dehusked rice seeds (cultivar IET-1991) was pre-soaked in distilled water for 2 h before treatment with Argemone oil for 16 h. A second set
192 of seeds, immersed in distilled water for 18 h, served as control. These treatments were repeated four times under identical conditions on different days. In a second experiment, two sets of dehusked seeds were irradiated with gamma rays at 20 kR; one set was soaked in H20 for 18 h, the second set was soaked in HsO for 2 h and then immersed in Argemone oil for 16 h. In yet another experiment two sets of dehusked seeds were immersed in HsO (18 h) or oil (2 h H20 + 16 h oil) before irradiation with 20 kR gamma rays. After completion of the treatment, the excess oil was removed from seeds which were then washed thoroughly in water for about 20 min. After ten days in petri dishes, germination counts were taken, and seedlings were transplanted into pots. The selfed panicles, at the rate of 250 to 300 per treatment, were collected from M1 plants. The Ms generation was raised in panicle progenies, and data on chlorophyll mutation and mutant frequencies were collected based on M~ segregating panicles and Ms mutant seedlings, respectively. The differences in the mutation and mutant frequencies were tested by the standard normal deviate (Z) test. Results and discussion As shown in Table I, a marked reduction in seed germination was induced by Argemone oil in comparison with seeds soaked in H20 (Expt. 1). A similar trend was observed for reduction in seed germination in combination treatments between gamma rays and Argemone oil compared with irradiated seeds soaked in HsO (Table I). The results obtained in the replicated Expt. 1 indicate that screw-pressed oil is a fairly potent mutagen; the mean chlorophyll mutation and mutant frequencies were 13.52 and 1.86%, respectively.
TABLE
I
FREQUENCY OF CHLOROPHYLL AND SEQUENTIAL TREATMENTS
Seed Get mio nation (%)
Treatment
Expt. 1 H20 18 h
MUTATIONS INDUCED BY ARGEMONE WITH GAMMA RAYS IN RICE
Total number of M 1 panicles analysed
Mutation frequency % -+ S.D.
--
OIL IN INDIVIDUAL
Total number of M2 seedlings scored
Mutant frequency % ± S.D.
94.8
1248
46,011
--
72.4
1158
1 3 . 5 2 +- 1 . 9 2
39,930
1 . 8 6 -+ 0 . 1 4 7
Expt. 2 20 kR + H20 18 h
82.5
263
1 0 . 5 9 -+ 1 . 6 2
8721
1 . 4 2 +- 0 . 1 0 2
20kR+ H20 + oil 1 6 h
H20
2 h + oil 1 6 h
2h 53.0
252
2 2 . 7 8 -+ 2 . 4 3 a
6192
3 . 7 3 -+ 0 , 1 7 2 a
Expt. 3 H20 18 h + 20 kR
64.5
261
1 8 . 4 6 -+ 2 . 2 1
7563
2.38 + 0.163
H20 2h+ oi116 h + 20 kR
39.5
250
2 9 . 1 7 -+ 2 . 9 3 b
5816
4 . 0 9 -+ 0 . 1 6 9 a
a Significant at P = 0 . 0 0 1 . b Significant at P = 0 . 0 1 .
193 In Expt. 2, significant increases in mutation and mutant frequencies were observed when irradiated seeds were immersed in oil as compared with watersoaked seeds. Similarly, the set of seeds pre-soaked in oil before irradiation showed significantly higher mutation and mutant frequencies compared with water pre-soaked seeds. In sequential treatments with oil and gamma rays both mutation and mutant frequencies were almost additive (Table I). The oil extracted from mustard seeds, containing a sufficient quantity of Argemone seeds, is known to cause erythema, epidemic dropsy, gastro-intestinal disturbances, hepatitis, oedema, cardiac failure, abortion, glaucoma and even cancer [2--9,11]. The toxic effects of oil were attributed to an alkaloid, namely sanguinarine, present in it [10]. Recent investigations have shown that a fatty acid factor is essential to potentiate the toxic effects of sanguinarine [6,7,9,11]. In L-cells, chromosomal aberrations were induced by treatment with sanguinarine [13]. Treatment of onion root tips with screw-pressed oil induced extensive chromosomal damage [12]. The observations made in this study prove conclusively that screw-pressed Argemone oil is a potent mutagen. The induction of mutations by Argemone oil in multicellular organisms has serious implications for the genetic hygiene of human beings, as it is being used for large scale adulteration of vegetable oils used as cooking media in India and elsewhere. Acknowledgements The authors gratefully acknowledge the encouragement given by Dr. O.S. Reddi and Dr. G.M. Reddy of the Department of Genetics, Osmania University. References 1 B h a t n a g a r , S.S., R . N . C h o p r a , B. P r a s a d , J . C . G o s h , M. Q u e r e s h i , M.N. S a h a , L. S r i r a m a n d B . L . M a n j u n a t h , T h e W e a l t h o f I n d i a , Vol. A - - B . , ( A d i c t i o n a r y of I n d i a n r a w m a t e r i a l s a n d i n d u s t r i a l p r o d ucts) published by the Council of Scientific and Industrial Research, 1948. 2 Lal, S.B., E p i d e m i c d r o p s y in B i h a r , I n d . Med. G a z . , 8 6 ( 1 9 5 1 ) 6 4 - - 7 1 . 3 Lal, R . B . a n d R . B . D a s g u p t a , I n v e s t i g a t i o n s i n t o the e p i d e m i o l o g y o f e p i d e m i c d r o p s y . X. A n o t e o n a n o u t b r e a k o f e p i d e m i c d r o p s y a s s o c i a t e d w i t h t h e use of m u s t a r d oil pressed f r o m seeds, a d u l t e r a t e d w i t h s e e d s o f A r g e m o n e m e x i c a n a , I n d . J o u r . Med. Res., 2 9 ( 1 9 4 1 ) 1 5 7 - - 1 6 5 . 4 L a l , R . B . a n d S.C. R o y , I n v e s t i g a t i o n i n t o the e p i d e m i o l o g y o f e p i d e m i c d r o p s y . I. I n t r o d u c t o r y n o t e s a n d h i s t o r i c a l s u r v e y , I n d . J o u r . Med. Res., 2 5 ( 1 9 3 7 ) 1 6 3 - - 1 7 6 . 5 P a t w a r d h a n , V . N . , in " N u t r i t i o n in I n d i a " p u b l i s h e d b y Dr. J . C . Patel, f o r I n d i a n J o u r n a l o f M e d i c a l Sciences (1952). 6 R a m a s h a s t r i , B . V . a n d S. B a b u , A r g e m o n e t o x i c i t y , A n n u a l r e p o r t , N a t i o n a l I n s t i t u t e o f N u t r i t i o n , H y d e r a b a d , 1 9 6 9 , p. 34. 7 R a m a s h a s t r i , B . V . a n d S. B a b u , A r g e m o n e t o x i c i t y , A n n u a l r e p o r t , N a t i o n a l I n s t i t u t e o f N u t r i t i o n , H y d e r a b a d , 1 9 7 0 , p. 31. 8 R a n a d i v e , K . J . , S . V . C o t h o s k a r a n d B.U. T e z a b w a l a , T e s t i n g c a r c i n o g e n i c i t y o f c o n t a m i n a n t s in edible oils. II. A r g e m o n e off in m u s t a r d oil, I n d . J o u r . Med. Res., 61 ( 1 9 7 3 ) 4 2 8 - - 4 3 4 . 9 R u k m i n i , C., S a n g u i n a r i n e p o t e n t i a t i n g f a c t o r in A r g e m o n e oil, I n d . J o u r . Med. Res., 59 ( 1 9 7 1 ) 1676--1680. 1 0 S a r k a r , S.N., I s o l a t i o n f r o m A r g e m o n e oil o f d i h y d r o s a n g u i n a r i n e a n d s a n g u i n a r i n e : t o x i c i t y o f sanguinarine, Nature (London), 162 (1948) 265--266. 11 S h e n o l i k a z , L.S., A r g e m o n e t o x i c i t y , Annuv.l r e p o r t , N a t i o n a l I n s t i t u t e o f N u t r i t i o n , H y d e r a b a d , 1 9 7 1 , p. 49. 1 2 S u b r a m a n y a m , S.S., P. V e n k a t R e d d y , G . P r e m v e e r R e d d y a n d D w a r a k a n a t h , K . M u r t h y , C y t o l o g i e a l e f f e c t s o f A r g e m o n e oil o n t h e m i t o t i c cells o f A l l i u m cepa, P r o c . I n d . A c a d . Sci., 7 9 ( 1 9 7 4 ) 2 1 6 - 226. 1 3 Svejda, J . J . , S . R . D v e r a k a n d R . A d a m e k , C h a n g e s in L-cells after t r e a t m e n t w i t h s o m e ( i s o c h i n o l i n e ) a l k a l o i d s , Scr. M e d . ( B r n o ) , 4 2 ( 1 9 6 9 ) 2 9 1 - - 2 9 5 .
