5 Quality of Selected Fruits and Vegetables of North America Downloaded from pubs.acs.org by UNIV OF MASSACHUSETTS AMHERST on 09/25/18. For personal use only.
Flavonoids, Mutagens, and Citrus R. M. HOROWITZ U.S. Department of Agriculture, Agricultural Research Service, Fruit and Vegetable Chemistry Laboratory, Pasadena, CA 91106
Flavonoids--the venerable group of plant constituents so beloved by natural products chemists--are regarded by some students of the nutritional arts as beneficial, even "semi-essential" components of the diet. Whether they are truly beneficial is a question debated for decades and one that is no more likely to be solved in the foreseeable future than many other well-known and enigmatic problems in nutrition. If flavonoids have dubious credentials as vitamins or semiessential dietary factors, i t has at least been generally agreed that, as a group, they are benign at the levels found in foods or incorporated in diet supplements and pharmaceuticals. They contain neither nitrogen nor sulfur--elements often associated with toxicity--and are thought to be metabolized chiefly to carbon dioxide and aromatic acids. Perhaps more important is the fact that since flavonoids occur in all higher plants (as well as some mosses, liverworts, fungi and ferns) they are, and always have been, a common constituent of the diet. It has been e s t i mated that the "average" American daily diet contains about 1 gram of flavonoids (1). Even i f this estimate were high by a factor of 2 to 3, the intake of flavonoids would still be substantial. To summarize, flavonoids are ubiquitous; conventional wisdom tells us they are unlikely to be harmful and may even be good for us. A new aspect of these compounds has surfaced and it is one that w i l l surely be visible for a long time. It has been found that certain flavonoids are mutagenic when examined in the Ames test and other short-term in vitro tests for mutagenicity. This finding is significant because there is substantial evidence to indicate that many known carcinogens are mutagens or are converted to mutagens by metabolic processes occurring in the body. Conversely, there is reason to believe that many mutagens are carcinogens and may also contribute to various genetic abnormali t i e s . It is the purpose of this article to review the data on flavonoid mutagenicity and to discuss some of the implications.
This chapter not subject to U.S. copyright. Published 1981 American Chemical Society
44 The Salmonella
QUALITY OF
S E L E C T E D FRUITS AND
VEGETABLES
test f o r mutagenicity
The best known and most w i d e l y used of the in vitro t e s t s i s t h a t developed by Ames and co-workers (2-5). In t h i s procedure, one of s e v e r a l s p e c i a l l y c o n s t r u c t e d h i s t i d i n e - r e q u i r i n g mutants of Salmonella typhimurium i s added to a h i s t i d i n e - d e f i c i e n t growth medium contained i n a P e t r i d i s h . Since the r a t e of spontaneous r e v e r s i o n t o h i s t i d i n e independence i s low, only background growth i s observed. I f , i n a p a r a l l e l experiment, a mutagenic substance i s added to the p l a t e c o n t a i n i n g the Salmonella b a c t e r i a and the growth medium, mutations l e a d i n g to h i s t i d i n e independence should occur and growth should be observed. A f t e r s e v e r a l days a count i s made of the r e v e r t a n t c o l o n i e s , each of which i s made up of b a c t e r i a c o n t a i n i n g f u n c t i o n a l r a t h e r than d e f e c t i v e h i s t i d i n e genes and each of which i s descended from a back-mutated bacterium. The number of r e v e r t a n t c o l o n i e s per nanomole of mutagen added i s o f t e n taken as a measure of the mutagenic potency of the substance t e s t e d . Experience has shown that i n c r e a s i n g the amount of mutagen i n the t e s t almost always r e s u l t s i n a l i n e a r i n c r e a s e i n the number of r e v e r t a n t c o l o n i e s produced. In p r a c t i c e , the experiment o u t l i n e d above i s not e n t i r e l y s a t i s f a c t o r y : many known c a r c i n o g e n s — b e n z [ a ] p y r e n e i s one examp l e — f a i l t o show up as mutagens. The reason f o r the f a i l u r e i s t h a t the compound i n q u e s t i o n happens not to be the proximate mutagen; i t becomes a mutagen only a f t e r i t e n t e r s the body and i s a l t e r e d by m e t a b o l i c processes. In order to simulate these metabolic processes in vitro and thereby improve the r e s u l t s i n the Salmonella t e s t , Ames has recommended the a d d i t i o n of v a r i o u s a c t i v a t i n g agents t o the t e s t p l a t e . Among these a c t i v a t i n g agents are homogenized l i v e r (the S-9 f r a c t i o n of r a t l i v e r microsomes induced w i t h p o l y c h l o r i n a t e d b i p h e n y l ( A r o c l o r ) or phénobarbital) and " f e c a l a s e " (a c e l l - f r e e , g l y c o s i d a s e - c o n t a i n i n g e x t r a c t of f e c e s ) . Other g l y c o s i d a s e - c o n t a i n i n g p r e p a r a t i o n s have been made from c e l l - f r e e e x t r a c t s of r a t c e c a l contents ("cecalase") and from Aspergillus niger ("hesperidinase"). The l i v e r homogenates seem t o c a r r y out a v a r i e t y of o x i d a t i v e and h y d r o l y t i c f u n c t i o n s ; the other p r e p a r a t i o n s are l a r g e l y hydrol y t i c and t h e i r c h i e f use i s i n the t e s t i n g of g l y c o s i d e s where i t i s necessary t o remove the sugar components i n order t o o b t a i n a p o s i t i v e t e s t . C l e a r l y , i n j u d g i n g how w e l l the in vitro Salmonella t e s t mimics in vivo m u t a g e n i c i t y , much depends on the choice of a c t i v a t i n g agents. I t has been pointed out that some substances may r e q u i r e r e d u c t i v e a c t i v a t i o n or may be metabolized by organs other than l i v e r or by c e l l components other than microsomes ( 6 ) . In some i n s t a n c e s treatment w i t h S-9 l i v e r homogenate causes d e a c t i v a t i o n (7^, .8, 9) . I n t e r e s t i n g d i s c u s s i o n s and r e b u t t a l s on v a r i o u s aspects of the s u b j e c t have appeared (10, 11, 12).
5.
HOROWITZ
Flavonoids Mutagens and Citrus
Behavior of f l a v o n o i d s i n the Salmonella
45
test
S t a r t i n g i n 1977, papers d e a l i n g w i t h t h i s t o p i c have come from h a l f a dozen d i f f e r e n t l a b o r a t o r i e s (13-18). The amount o f data i s considerable and, where the r e s u l t s of v a r i o u s i n v e s t i gators o v e r l a p , there i s f a i r l y s u b s t a n t i a l agreement. The kinds of f l a v o n o i d s that have been s t u d i e d , i n f r e e or combined form, are the f l a v o n e s , i s o f l a v o n e s , f l a v o n o l s , flavanones, d i h y d r o chalcones, catechins and anthocyanins. Of these, only the f l a v o n o l s show any a p p r e c i a b l e mutagenicity but i t must be emphasized that not a l l f l a v o n o l s are mutagenic. Table I contains a summary of the r e s u l t s reported f o r c e r t a i n f l a v o n o l s and t h e i r g l y c o s i d e s , a l l of which are or are thought t o be n a t u r a l l y o c c u r r i n g . I t a l s o contains data f o r the pentamethyl and pentaâcetyl d e r i v a t i v e s of q u e r c e t i n , n e i t h e r of which occurs n a t u r a l l y , and f o r a s m a l l group of a c t u a l or postul a t e d m e t a b o l i t e s of q u e r c e t i n . The p r i n c i p a l f i n d i n g s are these: 1) F i s e t i n ( I ) , q u e r c e t i n ( 2 ) , rhamnetin (3), 5,7-diO-methylquercetin (4) and m y r i c e t i n (5) are a l l mutagenic even i n the absence of any a c t i v a t i n g agent. When they are exposed t o S-9 l i v e r homogenate t h e i r mutagenic potency i n c r e a s e s , sometimes as much as t e n f o l d . 2) F l a v o n o l s 6-18 are not mutagenic by themselves but become so when exposed to S-9 o r , i n the case of g l y c o s i d e s , to a g l y c o s i d e - h y d r o l y z i n g enzyme. ( M y r i c i t r i n (25) would d o u b t l e s s l y become a c t i v e i n the presence of a h y d r o l y t i c enzyme.) The remaining f l a v o n o l s (19-28), as w e l l as the known or p o s t u l a t e d m e t a b o l i t e s of q u e r c e t i n (29-33), are i n a c t i v e even i f t r e a t e d w i t h S-9. 3) Quercetin (2) i s e a s i l y the most a c t i v e f l a v o n o l i n the Salmonella t e s t , yet i t i s at l e a s t an order of magnitude l e s s potent than such f a m i l i a r mutagens as benz[a]pyrene and chrysene. Quercetin, galangin ( 6 ) , kaempferol (7) and rhamnetin (3) are more or l e s s a c t i v e i n s e v e r a l other in vitro t e s t s designed t o detect genetic t o x i c i t y . For example, q u e r c e t i n has been shown t o transform hamster embryo c e l l s (kaempferol d i d not) (20) ; t o induce gene conversion i n yeast and f r a m e s h i f t mutation i n E. coli (17); to induce chromosome a b e r r a t i o n s and s i s t e r chromatid exchanges i n c u l t u r e d human and Chinese hamster c e l l s (21) ; and t o give a p o s i t i v e response i n the mouse leukemic c e l l mutation assay (9) . In some of these t e s t s the compound was a c t i v e without a d d i t i o n of S-9 l i v e r homogenate, w h i l e i n others S-9 a c t u a l l y decreased a c t i v i t y . 4) A c t i v i t y i s r e s t r i c t e d to but does not n e c e s s a r i l y occur i n compounds c o n t a i n i n g a f r e e hydroxy group at p o s i t i o n 3. Most of the a c t i v e compounds have a f r e e hydroxy group at p o s i t i o n 5 as w e l l , but there are exceptions, e.g., f i s e t i n (l) and 5,7-di-O-methylquercetin ( 4 ) , both of which have weak but d e f i n i t e a c t i v i t y . A f r e e hydroxy group at p o s i t i o n 7 i s not essent i a l for activity.
Kaempferol 3 , 5 , 7 , 4 - t e t r a OH
7
,
Galangin 3 , 5 , 7 - t r i OH
activated
6
only if
Myricetin 3,5,7,3»,4',5'-hexa OH
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5
5,7-Di-O-methylquercetin
4
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3
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2
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Mutagenic
Tester
Investigator
Table I . The Mutagenicity
2.4
3.2
0.17 1.0
0.25 0.63
0.25 2.6
2.8 12.7
0 0
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1.0
2.1
0.16 1.1
1.6 3.6
2.0 6.9
0.30 0.43
100
MacGregor (16)
2.0
11.6
0.14
98
7.3
0.12
100
Brown (18)
0.45
1537
3.9
3.8
5.3
0.12
98
0.68
1.6
0.35
0.35
100
4.09
0.54 1.08
0.79 2.27
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0 0
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98 100 1535 1538
98
in the
Bjeldanes (13)
Substances
Hardigree (17)
and Related
Sugimura (15)
of Some Naturally Occurring Flavonols Salmonella Test
5.
HOROWITZ
47
Flavonoids Mutagens and Citrus
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3-0-Methylquercetin
Caryatin
21
22
23
1
28
f
Robinet i n 3,7,3 ,4 ,5 -penta OH M y r i c i t r i n (myricetin 3-rhamnoside)
27
f
Penta-0-methylquercetin
26
f
3,3 -Di-0-methylquercetin
T
3,7-Di-0-methylquercetin
7 , 3 4 - t r i OH-3,5-di MeO
25
24
3-0-Methylgalangin 5,7-di OH-3-MeO
20
f
3,7-Dihydroxyflavone
TA no.
19
Non-mutagenic
Tester strain
Investigator
0
0
0
0
0
0
98
b
100
MacGregor (16)
Table
98
100
Brown (18) 1537
I-Continued
98
100
Sugimura (15) 98
Hardigree (17) 98 100 1535
1538
Bj eldanes (13)
Caffeic acid
Phloroglucinol
Phloroglucinol acid
31
32
33
0
0
metabolites 0 0
0 0
0
0
"Activated by c e c a l c e l l - f r e e e x t r a c t .
^ C a l c u l a t e d from data i n the o r i g i n a l paper; the l a r g e s t v a l u e was s e l e c t e d where more than one experiment was r e p o r t e d .
F i g u r e s represent number of r e v e r t a n t s per nanomole of compound t e s t e d ; data i n i t a l i c s are from experiments i n which no a c t i v a t i n g agent was used; S-9 l i v e r homogenate was used i n a l l other experiments except as noted; v a l u e s l e s s than 0.1 are l i s t e d as 0; TA98, TA1537 and TA1538 are f r a m e s h i f t t e s t e r s t r a i n s ; TA100 and TA1535 are b a s e - p a i r s u b s t i t u t i o n t e s t e r s t r a i n s ; f l a v o n o l numbering system i s shown i n s t r u c t u r e 2.
carboxylic
3,4-Dihydroxyphenylacetic acid
30
acid
or postulated
3-Hydroxyphenylacetic
metabolites
29
Quercetin
50
QUALITY
OF
SELECTED
FRUITS
AND
VEGETABLES
5) The s i t u a t i o n w i t h respect to s u b s t i t u t i o n i n the B - r i n g i s not e n t i r e l y c l e a r . MacGregor and Jurd (16), who s t u d i e d a l a r g e s e r i e s of compounds and have attempted to r a t i o n a l i z e s t r u c t u r e - a c t i v i t y r e l a t i o n s , suggest that the B - r i n g must be s u b s t i t u t e d i n a way that allows i t to become o x i d i z e d to a q u i nonoid intermediate. Thus, q u e r c e t i n could a u t o x i d i z e or be o x i d i z e d by the l i v e r microsomal system to the quinone 34a or the tautomeric quinone methide 34b. Other 3 , 4 * - d i h y d r o x y f l a v o n o l s would behave s i m i l a r l y . Monosubstituted d e r i v a t i v e s could be ortho-hydroxylated by the l i v e r p r e p a r a t i o n and methoxyl groups would presumably be demethylated at some stage. The mutagenicity of the f l a v o n o l s i s a t t r i b u t e d to the f a c t that the d e r i v e d q u i none methides are a l k y l a t i n g agents. The i d e a that a quinone methide or an analogous quinone s t r u c t u r e i s the proximate mutagen r e c e i v e s some support from a recent f i n d i n g that quinone methides such as 35 (derived from the corresponding f l a v y l i u m s a l t ) are mutagenic i n the Salmonella t e s t (22). f
I t appears that no attempts have been made to determine how l i v e r homogenate a l t e r s f l a v o n o l s , although t h i s i s a fundamental p o i n t and one that should be amenable to experiment. I t i s , n e v e r t h e l e s s , i n d i s p u t a b l e that c e r t a i n f l a v o n o l s are mutagenic i n c e r t a i n in vitro t e s t systems. Obviously, the question a r i s e s whether they c o n s t i t u t e a r i s k i n the d i e t of humans. Some authors are noncommittal on t h i s important p o i n t ; others have suggested t h a t c o n s i d e r a t i o n be given to a breeding program aimed at e l i m i n a t i n g or at l e a s t reducing the amount of f l a v o n o l s i n food p l a n t s (4^, 19) . This suggestion i s probably unworkable, e s p e c i a l l y s i n c e we do not even know to what extent f l a v o n o l s and t h e i r g l y c o s i d e s are e s s e n t i a l to the economy of the p l a n t . Moreover, a s u c c e s s f u l outcome would presumably do away w i t h what are thought t o be the b e n e f i c i a l e f f e c t s of f l a v o n o l s , i . e . , prot e c t i o n of l i p i d s and a s c o r b i c a c i d from o x i d a t i o n . In the sect i o n s below we d i s c u s s some f a c t o r s that may be u s e f u l i n e v a l u a t i n g the p o s s i b l e r i s k of i n g e s t i n g these compounds. F l a v o n o l occurrence and metabolism E x t r a p o l a t i o n of the r e s u l t s obtained from surveys of l e a f and flower p e t a l c o n s t i t u e n t s has l e d t o the c o n c l u s i o n that p r a c t i c a l l y a l l higher p l a n t s are able to synthesize f l a v o n o l s and that p l a n t t i s s u e s t o t a l l y l a c k i n g i n these substances are r a r e (23). Reported absences may i n most cases simply i n d i c a t e t h a t i n s u f f i c i e n t l y s e n s i t i v e methods were used. Quercetin and kaempferol are by f a r the most common f l a v o n o l s and they are almost always found i n the form of g l y c o s i d e s . In g e n e r a l , l e a v e s , f l o w e r s , f r u i t and other l i v i n g t i s s u e s c o n t a i n g l y c o s i d e s ; woody t i s s u e s tend to c o n t a i n aglycones and seeds may cont a i n e i t h e r . Because l i g h t i s r e q u i r e d i n the b i o s y n t h e t i c process, f l a v o n o l g l y c o s i d e s are u s u a l l y found i n highest concentra-
5.
HOROWITZ
Flavonoids Mutagens and Citrus
51
52
QUALITY
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FRUITS
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VEGETABLES
t i o n i n the s k i n s and outer leaves of f r u i t s and vegetables and i n lowest c o n c e n t r a t i o n i n root vegetables and other underground p a r t s of p l a n t s . In a recent review (24), g l y c o s i d e s of querc e t i n or kaempferol were reported to occur i n every one of a group of about 40 d i f f e r e n t food p l a n t s comprising f r u i t s , b e r r i e s and vegetables of a l l types. Thus, i t appears that the i n g e s t i o n of some q u a n t i t y of q u e r c e t i n and kaempferol g l y c o s i d e s i s unavoidable, w h i l e the i n g e s t i o n of any q u a n t i t y of q u e r c e t i n and kaempferol aglycones would be unusual. Since aglycones but not g l y c o s i d e s are mutagenic, we may w e l l wonder whether g l y c o s i d e s are hydrolyzed to aglycones a f t e r i n g e s t i o n . The u s u a l answer to t h i s question i s that h y d r o l y s i s does occur and the u s u a l evidence i s that c e c a l or f e c a l microf l o r a cause s p l i t t i n g of the sugars of p l a n t g l y c o s i d e s when t e s t e d in vitro. I t i s , however, by no means c e r t a i n that f l a v o noids i n v a r i a b l y undergo h y d r o l y s i s to the aglycone o r , i f they do, t h a t the aglycone p e r s i s t s f o r an a p p r e c i a b l e length of time. In a paper d e a l i n g w i t h the e f f e c t s of i s o l a t e d r a t c e c a l microf l o r a on f l a v o n o i d g l y c o s i d e s and other compounds, S c h e l i n e (25) pointed out t h a t only some of the r e a c t i o n s o c c u r r i n g i n the gut are revealed by in vitro experiments, s i n c e perhaps the m a j o r i t y of microorganisms growing i n the gut r e q u i r e s p e c i a l c o n d i t i o n s f o r t h e i r c u l t u r e . Moreover, the f a c t that a r e a c t i o n occurs i n an incubate may not a c c u r a t e l y r e f l e c t i t s q u a n t i t a t i v e s i g n i f i cance i n the gut, because the growth of minor organisms may be favored under the a r t i f i c i a l c o n d i t i o n s of the experiment. In vitro s t u d i e s on a rumen microorganism, Butyrivibrio sp. C 3 , have shown (26) that i t a n a e r o b i c a l l y degrades r u t i n (ll) or q u e r c i t r i n (10), but not the f r e e aglycone. At low temperature (4°) querc e t i n accumulates i n the medium w h i l e at higher temperature (39°) the g l y c o s i d e s disappear and the aglycone f a i l s to accumulate. (The products are p h l o r o g l u c i n o l (32), carbon d i o x i d e , 3,4-dihydroxybenzaldehyde and 3,4-dihydroxyphenylacetic a c i d (30)). When q u e r c e t i n i s provided as the s u b s t r a t e , very l i t t l e i s used. I t i s thought t h a t the Butyrivibrio g l y c o s i d a s e s are i n t r a c e l l u l a r and the block i n the use of f r e e aglycone i s a r e s u l t of i t s i n s o l u b i l i t y and i n a b i l i t y to enter the c e l l . In an e a r l i e r study (27) i t was found that the molds Aspergillus flavus and A. niger produce e x t r a c e l l u l a r enzymes capable of degrading r u t i n ; the products are r u t i n o s e , p r o t o c a t e c h u i c a c i d , p h l o r o g l u c i n o l c a r b o x y l i c a c i d (33) and a p h l o r o g l u c i n o l c a r b o x y l i c a c i d - p r o t o c a t e c h u i c a c i d e s t e r . Since q u e r c e t i n i s not detected as an intermediate i n the degradation, i t was suggested that one p o s s i b l e mode of a c t i o n might be cleavage of the h e t e r o c y c l i c r i n g of the i n t a c t g l y c o s i d e r a t h e r than h y d r o l y s i s of the sugars. Quercetin (except p o s s i b l y i n t r a c e s ) has not been detected as a m e t a b o l i t e of r u t i n a f t e r feeding i t to r a t s , r a b b i t s , guinea p i g s and humans. The only metabolic products reported are 3,4dihydroxyphenylacetic a c i d (30), 3-methoxy-4-hydroxyphenyJacetic a c i d , 3-hydroxyphenylacetic a c i d (29) and methylcatechol g l u c u r o -
5.
HOROWITZ
53
Flavonoids Mutagens and Citrus
nide (28, 29, 30). The same m e t a b o l i t e s are found when q u e r c e t i n i s fed i n s t e a d of r u t i n . In experiments w i t h s m a l l (5 mg) o r a l doses of *C-labeled q u e r c e t i n none of the unchanged compound could be found e i t h e r i n the u r i n e or g a s t r o i n t e s t i n a l contents of r a t s (31). The g l y c o s i d e m y r i c i t r i n (28) y i e l d s 3,5-dihydroxyp h e n y l a c e t i c a c i d as the major product i n both u r i n e and feces a t a dose of 100 mg/rat; some m y r i c e t i n (5) i s found i n the u r i n e but none i n the feces (32). A study of the C - l a b e l e d flavanone g l y c o s i d e , neohesperidin, and i t s r e d u c t i o n product, the sweetener neohesperidin dihydrochalcone, has shown t h a t , although metabolism takes p l a c e , n e i t h e r of the corresponding aglycones, h e s p e r e t i n and h e s p e r e t i n dihydrochalcone, occurs i n f r e e or combined form i n the u r i n e of r a t s that have been fed these substances (33). In germ-free r a t s f l a v o n o i d s f a i l to undergo the r i n g f i s s i o n r e a c t i o n s that y i e l d p h e n y l a c e t i c a c i d s and r e l a t e d compounds (30, 32). I t seems reasonable to conclude that the metabolism of querc e t i n g l y c o s i d e s i s brought about l a r g e l y by the a c t i o n of i n t e s t i n a l m i c r o f l o r a and that more than one pathway i s i n v o l v e d . One of these may be i n i t i a l h y d r o l y s i s of the g l y c o s i d e to the a g l y cone and another may be a d i r e c t a t t a c k on the f l a v o n o l moiety of the i n t a c t g l y c o s i d e . In any event, the a v a i l a b l e data suggest t h a t the aglycone has only a f l e e t i n g e x i s t e n c e before i t i s f u r t h e r degraded to aromatic a c i d s and carbon d i o x i d e . il
1I+
Feeding s t u d i e s S e v e r a l c h r o n i c t o x i c i t y s t u d i e s on r u t i n , q u e r c i t r i n and q u e r c e t i n have been p u b l i s h e d . A b r i e f summary of the e x p e r i ments i s given i n Table I I . In the e a r l y work of Wilson (34) and Ambrose (35) no e v i dence was obtained that i n d i c a t e d any a p p r e c i a b l e t o x i c i t y of r u t i n , q u e r c i t r i n or q u e r c e t i n at a 1% dose l e v e l i n r a t s . Histopathology was c a r r i e d out i n these s t u d i e s but no d e t a i l e d summary of the r e s u l t s was p u b l i s h e d . In a recent paper S a i t o and co-workers (37) reported a longer (^2-year) study of querc e t i n i n mice at a 2% dose l e v e l . There was a f a i r l y h i g h i n c i dence of tumors i n both t r e a t e d and c o n t r o l animals and some of the tumors i n the t r e a t e d group were of unusual types. (The q u e r c e t i n i n t h i s experiment was obtained commercially and used without f u r t h e r p u r i f i c a t i o n . ) I t was concluded that there i s no i n d i c a t i o n that q u e r c e t i n i s a potent carcinogen. In c o n t r a s t , Pamucku and co-workers (36) r e c e n t l y published the r e s u l t s of a 1-year r a t study i n which a high i n c i d e n c e of i n t e s t i n a l and bladder tumors was found i n animals that were kept on a g r a i n d i e t c o n t a i n i n g 0.1% q u e r c e t i n . ( I t was claimed that the querc e t i n was more than 99% pure.) The r e s u l t s are a l l the more a s t o n i s h i n g because the dose was only about 1/10 that used i n the Ambrose experiment and 1/20 that used i n the S a i t o experiment. C l e a r l y , i t i s d i f f i c u l t t o r e c o n c i l e the d i s c r e p a n c i e s i n
Rats; 5
Rats; 18
Mice; 35-38
Ambrose (1952) (35)
Pamukcu (1980) (36)
S a i t o (1980) (37)
Quercetin 0.1%
Quercetin 1%
Quercitrin 1%
Rutin 1%
Compound ; highest dose
Experiments
Quercetin Basal 2% pellet d i e t CE-2
Grain
See b
See b
Purina Dog Chow
Diet
Toxicity
Quercitrin
842
406
410
410
400
No s i g n i f i c a n t d i f f e r e n c e i n i n c i d e n c e of tumors i n c o n t r o l and t r e a t e d groups; s e v e r a l unusual tumors i n t r e a t e d group
C o n t r o l s : no tumors Treated: 80% developed i n t e s t i n a l tumors; 20% developed bladder tumors
Growth, organ weights, blood chemistry and h i s t o p a t h o l o g y normal
Growth, organ weights, blood chemistry and h i s t o p a t h o l o g y normal
Casein 10%, corn meal 73%, l i n s e e d o i l meal 10%, a l f a l f a 2%, bone ash 1.5%, sodium c h l o r i d e 0.5%, cod l i v e r o i l 3%.
8 g
66 g
66 g
70 g
Results
Quercetin
Growth, organ weights, h i s t o p a t h o l o g y and r e p r o d u c t i o n normal
and
T o t a l amount Duration ingested i n days per animal
on Rutin,
Rough estimates c a l c u l a t e d from data given i n the o r i g i n a l papers.
Rats; 5
Ambrose (1952) (35)
a
Rats; 6
Wilson (1947) (34)
Investigator
Animals; maximum number per group
Table I I . Chronic
5.
HOROWITZ
Flavonoids Mutagens and Citrus
55
these s t u d i e s but f a c t o r s such as p u r i t y , s o l u b i l i t y and moisture g i v e r i s e t o worrisome questions. I f a g r a i n r a t i o n were i n a d v e r t e n t l y exposed t o moisture could mycotoxins be formed? What i s the e f f e c t of the presumably i n s o l u b l e , c r y s t a l l i n e q u e r c e t i n used i n a l l these experiments? M i c r o b i a l a c t i o n in vitro i s extremely slow on c r y s t a l l i n e q u e r c e t i n (26). In humans given 4 grams of q u e r c e t i n o r a l l y more than h a l f of the dose i s excreted unchanged and there i s no evidence of a p p r e c i a b l e a b s o r p t i o n from the gut (38). The feeding s t u d i e s do not r e a l l y t e l l us whether q u e r c e t i n i s a carcinogen, although the balance of evidence suggests i t i s not. I t seems c l e a r that much of the e f f o r t has gone i n t o the wrong compound. R u t i n or some other n a t u r a l l y o c c u r r i n g g l y c o s i d e of q u e r c e t i n would be more l i k e l y than q u e r c e t i n i t s e l f t o g i v e a r e l i a b l e answer to the question of p o s s i b l e c a r c i n o g e n i c f l a v o n o l s i n foods of p l a n t o r i g i n . Flavonoids i n
Citrus
C i t r u s f r u i t i s a major source of f l a v o n o i d s i n the human d i e t ; the p e e l i s a major source of the f l a v o n o i d s that f i n d t h e i r way i n t o d i e t supplements and pharmaceuticals. ( I t may be noted, i n c i d e n t a l l y , that e i t h e r wet or d r i e d p e e l i s used as c a t t l e feed.) Flavanones, f l a v o n e s , f l a v o n o l s , dihydrochalcones and anthocyanins are the main types of f l a v o n o i d s i n Citrus (39). They occur i n highest c o n c e n t r a t i o n i n the p e e l and t o a l e s s e r extent i n the j u i c e and e d i b l e part of the f r u i t . By f a r the most abundant f l a v o n o i d s i n Citrus are flavanone g l y c o s i d e s , e.g., h e s p e r i d i n i n oranges and lemons, e r i o c i t r i n i n lemons, and n a r i n g i n and p o n c i r i n i n g r a p e f r u i t . H e s p e r i d i n and n a r i n g i n are e a s i l y i s o l a t e d and are s o l d commercially, the l a t t e r f o r use as a b i t t e r i n g agent. Both compounds can serve as s t a r t i n g mater i a l s f o r the manufacture of v a r i o u s dihydrochalcone sweeteners. Flavones are numerous and some of them, such as diosmin i n lemons or r h o i f o l i n i n g r a p e f r u i t , are present i n s u b s t a n t i a l q u a n t i t y . They occur e i t h e r as g l y c o s i d e s , C - g l y c o s y l d e r i v a t i v e s or permethyl ether d e r i v a t i v e s . Dihydrochalcones have thus f a r been found only i n kumquats and only i n the form of C - g l y c o s y l d e r i v a t i v e s . Anthocyanins occur c h i e f l y as c o n s t i t u e n t s of the blood oranges to which they c o n t r i b u t e the c h a r a c t e r i s t i c c o l o r . F l a v o n o l s , the group of f l a v o n o i d s most l i k e l y to c o n t a i n mutagens, are represented i n Citrus by a s m a l l number of examples. These i n c l u d e r u t i n (11), p o s s i b l y other g l y c o s i d e s of q u e r c e t i n (2), a g l y c o s i d e of kaempferol (7) and a g l y c o s i d e of isorhamnetin (13). With the exception of r u t i n , which has been obtained i n 3% y i e l d from the green f r u i t of the Satsumelo (a h y b r i d of grapef r u i t and t a n g e r i n e ) , a l l of these presumably promutagenic compounds are r e l a t i v e l y minor c o n s t i t u e n t s . Few q u a n t i t a t i v e s t u d i e s have been p u b l i s h e d , but i t has been estimated t h a t , a f t e r enzymatic h y d r o l y s i s of the n a t u r a l g l y c o s i d e s , 100 ml of s i n g l e -
56
QUALITY
OF
SELECTED
FRUITS
AND
VEGETABLES
s t r e n g t h lemon j u i c e contains about 2.2 mg of q u e r c e t i n i n a d d i t i o n to 20 mg of e r i o d i c t y o l and 1.4 mg of h e s p e r e t i n , both of which are flavanones (40). Other f l a v o n o l g l y c o s i d e s i n Citrus are the 3-£-D-glucosides of l i m o c i t r i n , l i m o c i t r o l and i s o l i m o c i t r o l . Nothing i s known about the p o s s i b l e mutagenicity of these minor g l y c o s i d e s or t h e i r aglycones. C i t r u s f r u i t s i n v a r i a b l y c o n t a i n complex a r r a y s of f l a v o n o i d compounds, the most important of which, both i n number and quant i t y , are flavanones and f l a v o n e s . A t o t a l of about 35 flavanones and f l a v o n e s have been i d e n t i f i e d , compared to about 6 f l a v o n o l s (39). Since flavanones and f l a v o n e s are not mutagens, the mutagenic p o t e n t i a l of crude, hydrolyzed p r e p a r a t i o n s of c i t r u s f l a vonoids i s l i k e l y to be s m a l l . Moreover, there are i n d i c a t i o n s t h a t the mutagenicity of f l a v o n o l s may be m o d i f i e d by the presence of other f l a v o n o i d s . Thus, Brown and D i e t r i c h (18) showed that the a c t i v i t y of g a l a n g i n (6) i n the Salmonella test i s strongly suppressed by the a d d i t i o n of e i t h e r the f l a v o n e a p i g e n i n or the f l a v o n o l r o b i n e t i n (27), although n e i t h e r of these compounds was more than m i n i m a l l y e f f e c t i v e i n reducing q u e r c e t i n m u t a g e n i c i t y . Perhaps the most thoroughly t e s t e d of a l l f l a v o n o i d s i s the sweetener neohesperidin dihydrochalcone, which i s formed by reducing the flavanone g l y c o s i d e n e o h e s p e r i d i n , a c o n s t i t u e n t of S e v i l l e oranges. The dihydrochalcone, a f t e r 2-year feeding t e s t s at v a r i o u s dose l e v e l s i n r a t s and dogs, gave no evidence of tumor i n d u c t i o n or t e r a t o g e n i c i t y (41). The compound has a l s o been checked (a) i n mice to see whether i t causes an i n c r e a s e i n the normal frequency of micronucleated polychromatic e r y t h r o c y t e s i n bone marrow (42) and (b) i n Salmonella t e s t e r s t r a i n s TA98, TA100, TA1535, TA1536, TA1537 and TA1538 to see whether i t causes r e v e r s i o n to h i s t i d i n e independence (14, 16, 18, 4^2, 43). Again, there was no evidence of mutagenicity. Are f l a v o n o l s a menace? Red wine (but not w h i t e ) , b l a c k tea and green t e a (but not c o f f e e ) , p i c k l e s and g r a p e j u i c e — a l l of these, we are warned, are mutagenic i n the Salmonella t e s t (5_, 44) . Some of them are a c t i v e even without h y d r o l y s i s . We are t o l d that " g l y c o s i d e s of querc e t i n , a mutagenic f l a v o n o i d , are present i n c o n s i d e r a b l e amounts i n our d i e t from a v a r i e t y of sources and, by means of h y d r o l y s i s , b a c t e r i a i n the human gut r e a d i l y l i b e r a t e the mutagen (_3). The i m p l i c a t i o n seems c l e a r enough: foods and beverages of p l a n t o r i g i n may c o n c e i v a b l y be a hazard to h e a l t h . Before acc e p t i n g t h i s view l e t us summarize what has been discussed above. The n a t u r a l s t a t e of f l a v o n o l s i n foods i s almost e x c l u s i v e l y a nonmutagenic g l y c o s i d e . H y d r o l y s i s l i b e r a t e s the f r e e f l a v o n o l s , some of which are mutagenic. Although c e r t a i n gut microf l o r a are able to cleave g l y c o s i d e s to aglycones, other pathways may a l s o be o p e r a t i v e whereby the aglycone moiety of the i n t a c t g l y c o s i d e i s degraded d i r e c t l y without the f r e e aglycone ever 11
5. HOROWITZ Flavonoids Mutagens and Citrus
57
being l i b e r a t e d . I n any event, s t u d i e s of the metabolism of q u e r c e t i n g l y c o s i d e have, thus f a r , provided no evidence f o r the accumulation of f r e e q u e r c e t i n ; i n s t e a d , non-mutagenic aromatic a c i d s r e s u l t i n g from the degradation of q u e r c e t i n are found. When f r e e q u e r c e t i n i s fed t o humans no d e t e c t a b l e q u a n t i t y i s absorbed through the gut; most of the q u e r c e t i n passes through unchanged and the r e s t presumably undergoes b a c t e r i a l degradation. Animal feeding s t u d i e s on q u e r c e t i n g l y c o s i d e s have given no evidence of c a r c i n o g e n i c i t y ; feeding s t u d i e s of q u e r c e t i n i t s e l f have given c o n f l i c t i n g r e s u l t s . F i n a l l y , on a more s p e c u l a t i v e l e v e l , C a i r n s (45) has noted that "presumably there has always been strong s e l e c t i o n pressure f o r adequate methods of DNA r e p a i r , and so i t i s h a r d l y s u r p r i s i n g t o f i n d such low l e v e l s of r i s k from agents l i k e UV l i g h t and X-rays, which have not changed i n i n t e n s i t y f o r m i l l i o n s of years." Since humans have always been exposed t o f l a v o n o l p r e c u r s o r s , i s i t not reasonable t o assume t h a t here too we are not l i k e l y t o exceed our DNA r e p a i r capabilities? More i n f o r m a t i o n on these complex subjects w i l l d o u b t l e s s l y be forthcoming but, on the b a s i s of what we know now, i t i s d i f f i c u l t t o reach any c o n c l u s i o n other than that the r i s k s a s s o c i a t e d w i t h the i n g e s t i o n of f l a v o n o l s are minimal. Addendum A f t e r t h i s a r t i c l e was submitted f o r p u b l i c a t i o n the r e s u l t s of a new feeding study were reported by Hirono and co-workers (46). I n one experiment ACI r a t s were kept f o r 540 days on b a s a l p e l l e t d i e t CE-2 c o n t a i n i n g e i t h e r 1% q u e r c e t i n , 5% q u e r c e t i n or 5% r u t i n ; i n a second experiment ACI r a t s were given e i t h e r 10% q u e r c e t i n or 10% r u t i n f o r 850 days. A t o t a l of 256 animals were used ( i n c l u d i n g c o n t r o l s ) . Most tumors found i n the experimental groups were a l s o present i n the corresponding c o n t r o l groups and there was no s i g n i f i c a n t d i f f e r e n c e between the i n c i d e n c e of tumors i n the c o n t r o l and experimental groups. The authors concluded that q u e r c e t i n and r u t i n are not c a r c i n o g e n i c i n ACI r a t s .
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Kühnau, J. World Review of Nutrition and Dietetics 1976, 24, 117-191. 2. Ames, B. N . ; McCann, J.; Yamasaki, E. Mutation Research 1975, 31, 347-364. 3. Ames, Β. N. Science 1979, 204, 587-593. 4. Ames, Bruce in "Genes, Cells and Behavior" edited by Ν. H. Horowitz and E. Hutchings, J r . ; W. H. Freeman and Company: San Francisco, 1980; pp 21-31.
58
5.
QUALITY OF SELECTED FRUITS AND VEGETABLES
Tamura, G.; Gold, C.; Ferro-Luzzi, Α.; Ames, Β. N. Proceedings of the National Academy of Sciences 1980, 77, 4961-4965. 6. Bridges, B. A. Nature 1976, 261, 195-200. 7. DeFlora, S. Nature 1978, 271, 455-456. 8. Aeschbacher, H. U . ; Chappins, C.; Wuerzner, H. P. Food and Cosmetics Toxicology 1980, 18, 605-613. 9. Meltz, M. L.; MacGregor, J. T. Mutation Research 1981, 88, 317-324. 10. Ashby, J.; Styles, J. A. Nature 1978, 261, 452-455. 11. Ames, Β. N . ; Hooper, K. Nature 1978, 274, 19-20. 12. Ashby, J.; Styles, J . A. Nature 1978, 274, 20-22. 13. Bjeldanes, L. F . ; Chang, G. W. Science 1977, 197, 577-578. 14. Brown, J. P.; Dietrich, P. S.; Brown, R. J. Biochemical Society Transactions 1977, 5, 1489-1492. 15. Sugimura, T.; Nagao, M.; Matsushima, T.; Yahagi, T.; Seino, Y . ; Shirai, Α.; Sawamura, M.; Natori, S.; Yoshihira, Κ.; Fukuoka, M.; Kuroyanagi, M. Proceedings of the Japan Academy, Ser. B. 1977, 53, 194-197. 16. MacGregor, J. T.; Jurd, L. Mutation Research 1978, 54, 297-309. 17. Hardigree, Α. Α.; Epler, J. L. Mutation Research 1978, 58, 231-239. 18. Brown, J. P.; Dietrich, P. S. Mutation Research 1979, 66, 223-240. 19. Brown, J. P. Mutation Research 1980, 75, 243-277. 20. Umezawa, K.; Matsushima, T.; Sugimura, T.; Hirakawa, T; Tanaka, M.; Katoh, Y . ; Takayama, S. Toxicology Letters 1977, 1, 175-178. 21. Yoshida, Μ. Α.; Sasaki, M.; Sugimura, K.; Kawachi, T. Proceedings of the Japan Academy, Ser. B. 1980, 56, 443-447. 22. Sweeny, J. G.; Iacobucci, G. Α.; Brusick, D.; Jagannath, D. R. 181st Meeting of the American Chemical Society, Atlanta, Georgia, March 29-April 3, 1981; Abstract AGFD 24. 23. Harborne, J . B. "Comparative Biochemistry of the Flavonoids"; Academic Press: London and New York, 1967; p 110. 24. Herrmann, K. Journal of Food Technology 1976, 11, 433-448. 25. Scheline, R. R. Acta Pharmacologica et Toxicologica 1968, 26, 332-342. 26. Krishnamurty, H. G.; Cheng, K.-J.; Jones, G. Α.; Simpson, F. J.; Watkin, J. E. Canadian Journal of Microbiology 1970, 16, 759-767. 27. Westlake, D. W. S.; Talbot, G.; Blakley, E. R.; Simpson, F. J . Canadian Journal of Microbiology 1959, 5, 621-629. 28. Murray, C. W.; Booth, A. N . ; DeEds, F . ; Jones, F. T. Journal of the American Pharmaceutical Association, Scientific Edition 1954, 43, 361-364. 29. Booth, A. N . ; Murray, C. W.; Jones, F. T.; DeEds, F. Journal of Biological Chemistry 1956, 223, 251-257.
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42. 43. 44. 45. 46.
59
Griffiths, L. A. in "Topics in Flavonoid Chemistry and Biochemistry" edited by L. Farkas, M. Gábor and F. Kállay; Elsevier Scientific Publishing Company: Amsterdam, 1975; pp 201-213. Petrakis, P. L.; Kallianos, A. G.; Wender, S. H . ; Shetlar, M. R. Archives of Biochemistry and Biophysics 1959, 85, 264-271. Griffiths, L. Α.; Smith, G. E. Biochemical Journal 1972, 130, 141-151. Horowitz, R. M.; Gentili, B. Citrus Research Conference, U.S. Department of Agriculture, Pasadena, California, December 8, 1976; Abstracts of Papers, pp 8-9. Wilson, R. H . ; Mortarotti, T. G.; Doxtader, Ε. K. Proceedings of the Society for Experimental Biology and Medicine 1947, 64, 324-327. Ambrose, A. M.; Robbins, D. J.; DeEds, F. Journal of the American Pharmaceutical Association, Scientific Edition 1952, 41, 119-122. Pamukcu, A. M.; Yalciner, S.; Hatcher, J. F.; Bryan, G. T. Cancer Research 1980, 40, 3468-3472. Saito, D.; Shirai, Α.; Matsushima, T.; Sugimura, T.; Hirono, I. Teratogenesis, Carcinogenesis and Mutagenesis 1980, 1, 213-221. Gugler, R.; Leschik, M.; Dengler, H. J. European Journal of Clinical Pharmacology 1975, 9, 229-234. Horowitz, R. M.; Gentili, B. in "Citrus Science and Technology," volume 1 edited by S. Nagy, P. E. Shaw and M. K. Veldhuis; Avi Publishing Company: Westport, Connecticut, 1977; pp 397-426. Vandercook, C. E . ; Stephenson, R. G. Journal of Agricultural and Food Chemistry 1966, 14, 450-454. Gumbmann, M. R.; Gould, D. H . ; Robbins, D. J.; Booth, A. N. in "Sweeteners and Dental Caries" edited by J. H. Shaw and G. G. Roussos; Information Retrieval, Inc.: Washington, D.C., 1978; pp 301-310. MacGregor, J. T. Toxicology and Applied Pharmacology 1979, 48, A47. Batzinger, R. P.; Ou, S.-Y. L.; Bueding, E. Science 1977, 198, 944-946. Uyeta, M.; Taue, S.; Mazaki, M. Mutation Research 1981, 88, 233-240. Cairns, J. Nature 1981, 289, 353-357. Hirono, I.; Ueno, I.; Hosaka, S.; Takanishi, H . ; Matsushima, T.; Sugimura, T.; Natori, S. Cancer Letters 1981, 13, 15-21.
RECEIVED
June
26,
1981.
