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Mutagenicities of 4-hydroxy-1,4-benzoxazinones naturally occurring in maize plants and of related compounds
191
Mutation Research, 66 (1979) 191--194 © Elsevier/North-HoUand Biomedical Press
Short Communication MUTAGENICITIES OF 4-HYDROXY-1,4-BENZOXAZINONES NATURALLY OCCURRING IN MAIZE PLANTS AND OF RELATED COMPOUNDS
Y. HASHIMOTO, K. SHUDO and T. OKAMOTO Faculty of Pharmaceutical Science, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113 (Japan) and
M. NAGAO, Y. TAKAHASHI and T. SUGIMURA
Biochemistry Division, National Cancer Center Research Institute, Tsukiji 5-1-1, Chuo-ku, Tokyo 104 (Japan) (Received 5 June 1978) (Accepted 22 August 1978)
The mutagenicities of naturally occurring substances in plants are now receiving attention [9]. This paper reports the mutagenicities of 4-hydroxy-l,4benzoxazinones and related compounds. 2,4-Dihydroxy-l,4-benzoxazinone (I) and 2,4-dihydroxy-7-methoxy-l,4-benzoxazinone (II) have been identified as insectistats and antifungal substances. These compounds occur naturally in several cereal plants, such as maize, wheat and rye [5,7,10], although it is unknown whether they are constituents of the grains. Their electrophilic activities [3], mentioned in relation to their antifungal actions, suggested that they might be mutagenic, and in fact, in this work we found that they had moderately strong mutagenicities. Thus, these compounds should not be overlooked during any consideration of naturally occurring mutagens or carcinogens in the environment. Materials and methods
Chemicals. T w o naturally occurring benzoxazinones (I and II) and two methoxy derivatives, 2-methoxy-4-hydroxy-l,4-benzoxazinone (III) and 2,7
192 TABLE
1
STRUCTURES
AND
BENZOMAZINONES
MELTING ADN
POINTS
EIGHT
OF NATURALLY
SYNTHETIC
OCCURRING
4-HYDROXY-
ANALOGS
m.p. 155-156 2,4-Dihydroxy-l,4-benzoxazinone ~'~N~-~-O OH
~
~.~o~-1~°-~o.
m.p. 162-163 2,4-Dihydroxy-7-methoxy-l,4-benzoxazinone
~ " "~/~ ~'O OH
m.p. 129-130 2-Methoxy-4-hydroxy-l,4-benzoxazinone ~"-.N/-.~O OH
IV
CH30~"~OTocH3
m.p. 148-150 2,7-Dimethoxy-4-hydroxy-l,4-benzoxazinone
OH
v
~°,~ °
m.p. 167-169 4-Hydroxy-l,4-benzoxazinone
o. v~
~o~o~
m.p. 125-127 4-Hydroxy-7-methoxy-l,4-benzoxazinone OH
m.p. 147
4-Hydroxy-6-methoxy-l,4-benzoxazinone
d.p. 197
4-Hydroxy-6-chloro-l,4-benzoxazinone
o.
cr
~
°
OH
IX
X
1 ( ) ~ FCH~ ~ / ~ 0 OH
~"~0
m.p. 123-124 2-Dimethyl-l,4-benzoxazinone
m.p. 117-118 N-Hydroxy-3,4-dihydrocarbostyril
OH
Mutation system. The mutation test with Salmonella typhimurium TA100 and TA98 was carried out as described previously [11]. S-9 Mix was prepared from the liver of rats injected with polychlorinated biphenyl. Mixtures of the test substance, S-9 MIX and bacteria were pre-incubated for 20 min at 37°C before the addition of molten soft agar. Results and discussion Table 2 summarizes the data on 2 naturally occurring 4-hydroxybenzoxazinones and 8 synthetic analogs. Compounds I and II, which occur naturally,
193 TABLE
[2
MUTAGENICITIES OF NATURALLY AND EIGHT SYNTHETIC ANALOGS
OCCURRING
4-HYDROXYBENZOXAZINONES
Revertant/:pmole TAI00 Compound
No.
TA98
+S-9 Mix
-S-9 Mix
I
608
285
18
0
II
223
0
12
0
IK
0
0
0
0
IV
0
0
0
0
V
0
0
0
0
VI
25
94
448
160
150
0
0
0
V~I
0
0
6
0
IX
0
0
0
0
X
0
0
0
0
V~
+S-9 Mix
-S-9 Mix
and 4-hydroxy-7-methoxybenzoxazinone (VI) and 4-hydroxy-6-methoxybenzoxazinone (VII) were mutagenic. Compounds I and II were more mutagenic on T A 1 0 0 than on TA98, showing activity in the presence of S-9 Mix; in the absence of S-9 Mix, the bactericidal effect of I seemed to hide its mutagenic activity. The dose--response curves of I and II with T A 1 0 0 and TA98 are shown in Figs. 1 and 2. The activity of I is stronger than that of II. Removal of
b
[ 200G
1000[ a
200
b
r
"E ..9o o
8
1000
0.5
1.0 0.5 mg/plate
1.0
0.5
1.0 ~ mg/p~te
Fig. 1. Mutagenicity of 2,4-dihydroxy-l,4obenzoxazinone in (a) TA100, and (I)) TA98. e, w i t h So9 Mix; o w i t h o u t S-9 Mix. Fig. 2. Mutagenicity of 2 , 4 - d i h y d r o x y - 7 - m e t h o x y - l , 4 - b e n z o x a z i n o n e in (a) TA100, and (b) TA98. e, w i t h S-9 Mix; o w i t h o u t S-9 Mix.
194
~I000
?
8
g ~
50C
a
o'.~
1'.o
o'.s
1'.o
mg/plate Fig. 3. Mutagenicity o f 4 - h y d r o x y - 7 - m e t h o x y - l , 4 - b e n z o x a z i n o n e in (a) T A I 0 0 , and (b) T A 9 8 . o, w i t h S-9 Mix; o, w i t h o u t S-9 Mix.
the 2+hydroxy group from I resulted in loss of mutagehic activity, because 4-hydroxy-l,4-benzoxazinone (V) is not mutagenic. On the other hand, VI without the 2-hydroxy group of II was active on TA98 in the presence of S-9 Mix (Fig. 3). This c o m p o u n d was only weakly active on T A 1 0 0 without 8-9 Mix. Thus the presence of either the 2-hydroxy group or the 7-methoxy group seems to be required for mutagenic activity. These two functional groups enhance the reactivity of the N--O bond [3,4]. Compound VII, with a 6-methoxy group, had weak mutagenic activity. The t w o 2 - m e t h o x y derivatives, III and IV, were also completely inactive, although the activity of compound IV was expected to be similar to that of VI. Compounds VIII, IX and X were non-mutagenic. The mechanism of the mutagenic activity of these hydroxamic acids is not clear. Most of these compounds required metabolic activation, and the pathway o f this metabolic activation requires further study. It is interesting that the structures of these compounds are similar to that of N-hydroxyphenacetin [8]. The naturally occurring compounds I and II have sufficiently high specific mutagenicities to be regarded as naturally occurring mutagens. Further studies are required on the significance of the mutagenicity of N-hydroxybenzoxazinones to animals and humans, and on the distributions of I and II and related compounds in plants and in animal foods. References 1 Courts, R . T . , D. N o b l e and D . G . W i b b e r l e y , S o m e cyclic h y d r o x a m i c acids, J. Pharm. Pharmacol., 16 (1964) 773--788. 2 C o u t t s , R . T . , a n d N.J. P o u n d , Preparation of an aromatic h y d r o x y l a m i n e and s o m e cyclic h y d r o x a m i c acids, and their reaction w i t h h y d r o c h l o r i c acid, Can. J. C h e m . , 48 ( 1 9 7 0 ) 1 8 5 9 - - 1 8 6 4 . 3 H a s h i m o t o , Y., T. Ohta, K. S h u d o and T. O k a m o t o , R e a c t i o n s o f 4 - h y d r o x y - 2 H - 1 , 4 - b e n z o x a z i n - 3 one-derivatives w i t h s o m e n u e l e o p h i l e s , H e t e r o c y c l e s , 9 ( 1 9 7 8 ) 1 0 9 . 4 H a s h i m o t o , Y., T. Ohta, K. S h u d o and T. O k a m o t o , u n p u b l i s h e d data. 5 H o f m a n , J., and O. H o f m a n o v e , 1 , 4 - B e n z o x a z i n e derivatives in plants, Eur. J. B i o c h e m . , 8 ( 1 9 6 9 ) 109--112. 6 J e r n o w , J.L., and R. R o s e n , U . S . Patent 3 , 8 6 2 , 1 8 0 , Jan. 21, 1 9 7 5 . 7 Klun, J.A., C.L. T i p s o n and T . A . Brindley, 2 , 4 - D i h y d r o x y - 7 - m e t h o x y - l , 4 - b e n z o x a z i n - 3 - o n e ( D I M B O A ) , an active agent in the resistance o f m a i z e to the Ettropean corn borer, J. E c o n . E n t o m o l . , 60 (1967) 1529--1532. 8 S h u d o , K., T. Ohta, Y. Orihara, T. O k a m o t o , M. Nagao, Y. T a k a h a s h i and T. Sugimura, Mutagenicities o f p h e n a c e t i n and its m e t a b o l i t e s , M u t a t i o n R e s . , 58 ( 1 9 7 8 ) 3 6 7 - - 3 7 0 . 9 S c h o e n t a l , R.0 Carcinogens in plants and m i c r o o r g a n i s m s , C h e m i c a l Carcinogens, A C S M o n o g r a p h 1 7 3 , A m . C h e m . S o c . , W a s h i n g t o n , 1 9 7 6 , pp. 6 2 6 - - 6 8 9 . 10 T i p s o n , C.L., J.A. Klun, R . R . Husted and M.D. Pierson, Cyclic h y d r o x a m i c acids and related c o m p o u n d s f r o m m a i z e . I s o l a t i o n and characterization, B i o c h e m i s t r y , 6 ( 1 9 6 7 ) 2 8 6 6 - - 2 8 7 0 . 11 Yahagi. T.. M. Nagao. Y . S e i n o . T. Matsushima. T. Su~imttra and M. Okada_ Muta~enieitv of/V-nitrn.
Mutation Research, 66 (1979) 191--194 © Elsevier/North-HoUand Biomedical Press
Short Communication MUTAGENICITIES OF 4-HYDROXY-1,4-BENZOXAZINONES NATURALLY OCCURRING IN MAIZE PLANTS AND OF RELATED COMPOUNDS
Y. HASHIMOTO, K. SHUDO and T. OKAMOTO Faculty of Pharmaceutical Science, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113 (Japan) and
M. NAGAO, Y. TAKAHASHI and T. SUGIMURA
Biochemistry Division, National Cancer Center Research Institute, Tsukiji 5-1-1, Chuo-ku, Tokyo 104 (Japan) (Received 5 June 1978) (Accepted 22 August 1978)
The mutagenicities of naturally occurring substances in plants are now receiving attention [9]. This paper reports the mutagenicities of 4-hydroxy-l,4benzoxazinones and related compounds. 2,4-Dihydroxy-l,4-benzoxazinone (I) and 2,4-dihydroxy-7-methoxy-l,4-benzoxazinone (II) have been identified as insectistats and antifungal substances. These compounds occur naturally in several cereal plants, such as maize, wheat and rye [5,7,10], although it is unknown whether they are constituents of the grains. Their electrophilic activities [3], mentioned in relation to their antifungal actions, suggested that they might be mutagenic, and in fact, in this work we found that they had moderately strong mutagenicities. Thus, these compounds should not be overlooked during any consideration of naturally occurring mutagens or carcinogens in the environment. Materials and methods
Chemicals. T w o naturally occurring benzoxazinones (I and II) and two methoxy derivatives, 2-methoxy-4-hydroxy-l,4-benzoxazinone (III) and 2,7
192 TABLE
1
STRUCTURES
AND
BENZOMAZINONES
MELTING ADN
POINTS
EIGHT
OF NATURALLY
SYNTHETIC
OCCURRING
4-HYDROXY-
ANALOGS
m.p. 155-156 2,4-Dihydroxy-l,4-benzoxazinone ~'~N~-~-O OH
~
~.~o~-1~°-~o.
m.p. 162-163 2,4-Dihydroxy-7-methoxy-l,4-benzoxazinone
~ " "~/~ ~'O OH
m.p. 129-130 2-Methoxy-4-hydroxy-l,4-benzoxazinone ~"-.N/-.~O OH
IV
CH30~"~OTocH3
m.p. 148-150 2,7-Dimethoxy-4-hydroxy-l,4-benzoxazinone
OH
v
~°,~ °
m.p. 167-169 4-Hydroxy-l,4-benzoxazinone
o. v~
~o~o~
m.p. 125-127 4-Hydroxy-7-methoxy-l,4-benzoxazinone OH
m.p. 147
4-Hydroxy-6-methoxy-l,4-benzoxazinone
d.p. 197
4-Hydroxy-6-chloro-l,4-benzoxazinone
o.
cr
~
°
OH
IX
X
1 ( ) ~ FCH~ ~ / ~ 0 OH
~"~0
m.p. 123-124 2-Dimethyl-l,4-benzoxazinone
m.p. 117-118 N-Hydroxy-3,4-dihydrocarbostyril
OH
Mutation system. The mutation test with Salmonella typhimurium TA100 and TA98 was carried out as described previously [11]. S-9 Mix was prepared from the liver of rats injected with polychlorinated biphenyl. Mixtures of the test substance, S-9 MIX and bacteria were pre-incubated for 20 min at 37°C before the addition of molten soft agar. Results and discussion Table 2 summarizes the data on 2 naturally occurring 4-hydroxybenzoxazinones and 8 synthetic analogs. Compounds I and II, which occur naturally,
193 TABLE
[2
MUTAGENICITIES OF NATURALLY AND EIGHT SYNTHETIC ANALOGS
OCCURRING
4-HYDROXYBENZOXAZINONES
Revertant/:pmole TAI00 Compound
No.
TA98
+S-9 Mix
-S-9 Mix
I
608
285
18
0
II
223
0
12
0
IK
0
0
0
0
IV
0
0
0
0
V
0
0
0
0
VI
25
94
448
160
150
0
0
0
V~I
0
0
6
0
IX
0
0
0
0
X
0
0
0
0
V~
+S-9 Mix
-S-9 Mix
and 4-hydroxy-7-methoxybenzoxazinone (VI) and 4-hydroxy-6-methoxybenzoxazinone (VII) were mutagenic. Compounds I and II were more mutagenic on T A 1 0 0 than on TA98, showing activity in the presence of S-9 Mix; in the absence of S-9 Mix, the bactericidal effect of I seemed to hide its mutagenic activity. The dose--response curves of I and II with T A 1 0 0 and TA98 are shown in Figs. 1 and 2. The activity of I is stronger than that of II. Removal of
b
[ 200G
1000[ a
200
b
r
"E ..9o o
8
1000
0.5
1.0 0.5 mg/plate
1.0
0.5
1.0 ~ mg/p~te
Fig. 1. Mutagenicity of 2,4-dihydroxy-l,4obenzoxazinone in (a) TA100, and (I)) TA98. e, w i t h So9 Mix; o w i t h o u t S-9 Mix. Fig. 2. Mutagenicity of 2 , 4 - d i h y d r o x y - 7 - m e t h o x y - l , 4 - b e n z o x a z i n o n e in (a) TA100, and (b) TA98. e, w i t h S-9 Mix; o w i t h o u t S-9 Mix.
194
~I000
?
8
g ~
50C
a
o'.~
1'.o
o'.s
1'.o
mg/plate Fig. 3. Mutagenicity o f 4 - h y d r o x y - 7 - m e t h o x y - l , 4 - b e n z o x a z i n o n e in (a) T A I 0 0 , and (b) T A 9 8 . o, w i t h S-9 Mix; o, w i t h o u t S-9 Mix.
the 2+hydroxy group from I resulted in loss of mutagehic activity, because 4-hydroxy-l,4-benzoxazinone (V) is not mutagenic. On the other hand, VI without the 2-hydroxy group of II was active on TA98 in the presence of S-9 Mix (Fig. 3). This c o m p o u n d was only weakly active on T A 1 0 0 without 8-9 Mix. Thus the presence of either the 2-hydroxy group or the 7-methoxy group seems to be required for mutagenic activity. These two functional groups enhance the reactivity of the N--O bond [3,4]. Compound VII, with a 6-methoxy group, had weak mutagenic activity. The t w o 2 - m e t h o x y derivatives, III and IV, were also completely inactive, although the activity of compound IV was expected to be similar to that of VI. Compounds VIII, IX and X were non-mutagenic. The mechanism of the mutagenic activity of these hydroxamic acids is not clear. Most of these compounds required metabolic activation, and the pathway o f this metabolic activation requires further study. It is interesting that the structures of these compounds are similar to that of N-hydroxyphenacetin [8]. The naturally occurring compounds I and II have sufficiently high specific mutagenicities to be regarded as naturally occurring mutagens. Further studies are required on the significance of the mutagenicity of N-hydroxybenzoxazinones to animals and humans, and on the distributions of I and II and related compounds in plants and in animal foods. References 1 Courts, R . T . , D. N o b l e and D . G . W i b b e r l e y , S o m e cyclic h y d r o x a m i c acids, J. Pharm. Pharmacol., 16 (1964) 773--788. 2 C o u t t s , R . T . , a n d N.J. P o u n d , Preparation of an aromatic h y d r o x y l a m i n e and s o m e cyclic h y d r o x a m i c acids, and their reaction w i t h h y d r o c h l o r i c acid, Can. J. C h e m . , 48 ( 1 9 7 0 ) 1 8 5 9 - - 1 8 6 4 . 3 H a s h i m o t o , Y., T. Ohta, K. S h u d o and T. O k a m o t o , R e a c t i o n s o f 4 - h y d r o x y - 2 H - 1 , 4 - b e n z o x a z i n - 3 one-derivatives w i t h s o m e n u e l e o p h i l e s , H e t e r o c y c l e s , 9 ( 1 9 7 8 ) 1 0 9 . 4 H a s h i m o t o , Y., T. Ohta, K. S h u d o and T. O k a m o t o , u n p u b l i s h e d data. 5 H o f m a n , J., and O. H o f m a n o v e , 1 , 4 - B e n z o x a z i n e derivatives in plants, Eur. J. B i o c h e m . , 8 ( 1 9 6 9 ) 109--112. 6 J e r n o w , J.L., and R. R o s e n , U . S . Patent 3 , 8 6 2 , 1 8 0 , Jan. 21, 1 9 7 5 . 7 Klun, J.A., C.L. T i p s o n and T . A . Brindley, 2 , 4 - D i h y d r o x y - 7 - m e t h o x y - l , 4 - b e n z o x a z i n - 3 - o n e ( D I M B O A ) , an active agent in the resistance o f m a i z e to the Ettropean corn borer, J. E c o n . E n t o m o l . , 60 (1967) 1529--1532. 8 S h u d o , K., T. Ohta, Y. Orihara, T. O k a m o t o , M. Nagao, Y. T a k a h a s h i and T. Sugimura, Mutagenicities o f p h e n a c e t i n and its m e t a b o l i t e s , M u t a t i o n R e s . , 58 ( 1 9 7 8 ) 3 6 7 - - 3 7 0 . 9 S c h o e n t a l , R.0 Carcinogens in plants and m i c r o o r g a n i s m s , C h e m i c a l Carcinogens, A C S M o n o g r a p h 1 7 3 , A m . C h e m . S o c . , W a s h i n g t o n , 1 9 7 6 , pp. 6 2 6 - - 6 8 9 . 10 T i p s o n , C.L., J.A. Klun, R . R . Husted and M.D. Pierson, Cyclic h y d r o x a m i c acids and related c o m p o u n d s f r o m m a i z e . I s o l a t i o n and characterization, B i o c h e m i s t r y , 6 ( 1 9 6 7 ) 2 8 6 6 - - 2 8 7 0 . 11 Yahagi. T.. M. Nagao. Y . S e i n o . T. Matsushima. T. Su~imttra and M. Okada_ Muta~enieitv of/V-nitrn.