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microsome test
Mutation Research, 124 (1983) 25-34
25
Elsevier MTR 00805
Mutagenicity of natural n a p h t h o q u i n o n e s and b e n z o q u i n o n e s in the S a l m o n e l l a / m i c r o s o m e test Leena Tikkanen 1,,, Taijiro Matsushima 1,**, Shinsaku Natori 2,*** and Kunitoshi Yoshihira 2 l Department of Molecular Oncology, Institute of Medical Science, Universityof Tokyo, Shirokanedai, Minato- ku, Tokyo 108; and 2 National Institute of Hygienic Sciences, Kamiyoga, Setagaya. ku, Tokyo 158 (Japan)
(Received2 March 1983) (Accepted 11 May 1983)
Summary The mutagenicities of naturally occurring naphthoquinones and benzoquinones were tested by the pre-incubation method with Salmonella typhimurium strains TA98, TA100 and TA2637, which all contain plasmid pKM101. 6 of the 16 naphthoquinones tested, i.e., plumbagin, naphthazarin, 2-hydroxynaphthoquinone, vitamin K 3 (menadione), juglone and 7-methyljuglone, were mutagenic to strain TA2637 with metabolic activation. Except for juglone and 7-methyljuglone, these compounds also had slight mutagenic effects on strain TA98 with $9 mix. All the mutagenic naphthoquinones contain one or two hydroxyl a n d / o r methyl substituents. The naphthoquinone mompain, which has four hydroxyl groups, was not mutagenic. Unsubstituted fl-naphthoquinone, naphthoquinones with a prenyl side chain and all bi-naphthoquinone derivatives tested were non-mutagenic. None of the 13 benzoquinones examined was mutagenic to any of the strains used with or without metabolic activation. These results show that natural naphthoquinones are mutagenic when they have only one or two hydroxyl a n d / o r methyl substituents.
Quinone pigments are a large class of naturally occurring coloring substances present in various families of plants and fungi. The largest group of naturally * Recipient of a scholarship from the Japanese government during the period of this work. Present address: TechnicalResearch Centre of Finland, Food Research Laboratory,Biologinkuja1, SF-02150 Espoo i 5, Finland. ** To whom requests for reprints should be addressed. *** Present address: Meiji Collegeof Pharmacy,Yato-cho,Tanashi-shi, Tokyo 188 (Japan). 0165-1218/83/$03.00 © 1983 ElsevierSciencePublishers B.V.
26 occurring quinones is that of anthraquinones, but naphthoquinones and benzoquinones are also common. Most naphthoquinones are plant products, whereas benzoquinones have been found in higher plants and in fungi (Thomson, 1971). Substituted 1,4-naphthoquinones occur in nature as various pigments and as K vitamins. Several of these compounds, such as lawsone (2-hydroxynaphthoquinone), alkannin and its antipode (shikonin), are used as dyestuffs. Juglone, methyljuglone and plumbagin are widely used in folk medicines (Thomson, 1971). K vitamins are widely distributed in higher green plants; they are used as prothrombogenic vitamins. Some hydroxybenzoquinones are widely distributed in Myrsinaceae plants (Thomson, 1971; Ogawa and Natori, 1968a, 1968b). Some of these benzoquinones are used for medical purposes because they have anthelminthic and purgative properties. Terphenyl derivatives of benzoquinones occur mainly in fungi as colored and toxic components (Thomson, 1971). Ubiquinones are a group of benzoquinones involved in electron transport in mitochondrial preparations. They are present in most aerobic organisms from bacteria to higher plants and animals. Although naphthoquinones and benzoquinones are widely distributed in nature and are extensively used by man for various purposes, there have been few reports about their toxicological properties, including their mutagenicities and carcinogenicities. In the present study, we examined the mutagenicities of natural naphthoquinones and benzoquinones by the pre-incubation method with Salmonella typhimurium strains TA98, TA100 and TA2637.
Materials and methods
Test compounds The naphthoquinones and benzoquinones tested, and their structures, sources and references to the methods used for their isolation are presented in Tables 1 and 2. The naphthoquinones naphthazarin, 2-hydroxynaphthoquinone,/3-naphthoquinone, vitamin K 3 (menadione) and vitamin K~ were purchased from Tokyo Kasei Kogyo Co., Tokyo. Plumbagin, juglone, 7-methyljuglone, mompain, eliptinone, mamegakinone, isodiospyrin, bisisodiospyrin and elsinochrome A were isolated at the National Institute of Hygienic Sciences, Tokyo. Alkannin was a generous gift from Dr. U. Sankawa, Faculty of Pharmaceutical Sciences, University of Tokyo, Tokyo. Tanshinone II was a generous gift from Dr. H. Kakisawa, Department of Chemistry, Tsukuba University, Ibaraki. The benzoquinone ubiquinone-9 was purchased from Taisho Pharmaceutical Co., Tokyo. Embelin, rapanone, maesaquinone, dihydromaesaquinone, rapanone monomethyl ether, rapanone dimethyl ether, helicobasidin, (+)-dihydroperezone, ardisiaquinone A, ardisiaquinone B, 2,5-di-p-anisyl-p-benzoquinone and 2,5-di-(pacetoxyphenyl)-3-acetoxy-p-benzoquinonewere isolated or prepared at the National Institute of Hygienic Sciences, Tokyo. The purities of the test compounds were confirmed by physical data and by thin-layer chromatography.
27
TABLE 1 STRUCTURES AND SOURCES OF NAPHTHOQUINONES TESTED NAME
STRUCTURE
SOURCE
REFERENCE (Isolation method)
o 1.
PLUMBAGIN
~M* No o
DlOspyros kaki
2.
NAPHTNAZARIN
~
3.
2-HYDROXYNAPHTHO- ~ o N QUINONE o
synthetic
4.
MENADIONE
@..
synthetic
5.
JUGLONE
oH~o
Juglsns regis
Ikekews, et el., 1967
6.
7-METHYLJUGLONE M e ~
Dlospyros lotus
Yoshihlra, et el., 1971 Tezuka, et el., 1973
Hollcobusldlum mompa
Netorl, et el.,
Tezuks, et el., 1972, 1973
oH o
synthetic o
o oH o oH o 7.
MOMPAIN
8.
fl-NAPHTHOQUINONE
9.
ELIPTINONE
~ Ho~OH o
cr;r ° o oH
10.
MAMEGAKINONE
11.
ISOOIOSPYRIN
12.
BISISOOIOSPYRIN
13.
ELSINOCHROME A
1967
synthetic oH o
Dlospyros maritime Me
Tezuke, et el., 1973
Me
OH o 0014 u * ~ ~ o'~ o ''~'Me Dloepyros lotus oH o ~ Me~o Me oM o
HO
Dloepyros lotus
OH 0
O HOe
OH
Yoshlhlra, et el., 1971 Tezuka, et el., 1973 Yoshlhlre, et el., 1971 Tezuks, et el., 1973
Yoshihire, et el., 1971 Tezuke, et el., 1973
Dloepyros lotus o[k O0uH 0M* ~ MOO COMI MoO~ O
Elslnoee erellee
Chen, et el., 1966
Mecrotomle euchrome
Senkawe, et el.0 1981
Salvia mlltlorrhlzs
Okumure, et el., 1961
O~ M COMe
O,H (~MeMe Me~U~OH
14. ALKANNIN
16.
TANSHINONE II
19.
VITAMIN K 1
~
@'" °7
°.:cH-(~ °.=°.:°.2~:.
c.:
synthetic
28 TABLE 2 S T R U C T U R E S A N D SOURCES OF B E N Z O Q U I N O N E S TESTED NAME
STRUCTURE
SOURCE
o•c
H
1. EMBELIN
H
11H23
HO
C13H27
2. RAPANONE
REFERENCE ( Isolation method)
Myrslne sequlnll
Ogawa and Natorl, 1968 a
Ardlsla crenata
Ogawe and Natorl, 1 9 6 8 a
Masse Japonica
Ogawa and Natorl, 1 9 6 5
partial synthesis
Ogawa and Natorl, 1 9 6 5
partial synthesis
O g a w l end Hatori, 1 9 6 8 b
partial synthesis
Ogawa and Netorl, 1 9 6 8 b
O 3. MAESAOUINONE H
H2113CH:CH-C4H 9
0 Me~)H
4. DIHYDROMAESAQUINONE
HO
5. RAPANONE MONOMETHYL ETHER
C19H39
Meo
c13~27 o
6. RAPANONE DIMETHYL ETHER
MeO
7. UBIQUINONE-9
O CIH3 MeO~U~(C H2CHIC-CH2)SH MIO" Y "M•
13H27
synthetic
0
""oH_
8. HELICOBASIDIN
H
9. (::~)-D|H YORO PEREZONE
Hellcobasldlum momba
Natorl, et el., 1 9 6 4
synthetic
Yamaguchi, 1942
Ardisia sleboldll
Ogawa and Natorl, 1 9 6 8 b
Ardlsla sleboldli
Ogawa end Nalorl, 1 9 6 8 b
~--- Oue
synthetic
Takahaahi, i t el., 1 9 7 6
OAc
synthetic
Tekahashl, et el., 1 9 7 6
Me Ma Me O Me
~ Me
10, ARDISIAOUINONE A
~l~ 10H
HO- y
(CH)
H
11. AROISIAOUINONE B
0
12. 2 , 5 - D I - p - A N I S Y L - p BENZOOUINONE
MeO.~--~
13. 2,5-DI-(p-ACETOXYPHENYL)-3-ACETOXY-pBENZOOUINONE
,
o 4
c
0
~
Bacterial strains Salmonella typhimurium strains TA100, TA2637 and TA98 were used as tester strains. These strains were derived from S. typhimurium strains TA1535, TA1537 and TA1538, respectively, by introduction of plasmid pKM101 (Ames et al., 1975). The tester strains were provided by Dr. B.N. Ames, University of California, Berkeley, California. They were grown overnight in nutrient broth (Difco) with shaking before use.
29 Mutation test
The Salmonella mutation test was performed by the pre-incubation method, a modification of the plate-test method (Yahagi et al., 1975). A mixture of the test compound in 50/~1 of dimethylsulfoxide, 0.5 ml of $9 mix or 0.2 M phosphate buffer (pH 7.4) and 100/~1 of culture of the tester strain was pre-incubated for 30 min at 30°C in a test-tube. Then 2 ml of top agar containing 0.5 mM histidine and biotin was added, and after thorough mixing, the solution was poured on to Vogel-Bonner minimal agar plates. The plates were incubated for 48 h at 37°C, and then revertant colonies were counted. Liver homogenate ($9) was prepared by the method of Ames et al. (1975) from the livers of rats treated with Na-phenobarbital and 5,6-benzoflavone (Matsushima et al., 1976). $9 mix contained (per ml) 100/~1 of $9, 4/tmoles each of NADPH and NADH, 5 /tmoles of glucose-6-phosphate, 33 /~moles of KC1, 8 ~tmoles of MgC12 and 100/~moles of Na-phosphate buffer, pH 7.4. The result was defined as positive when the compound induced a dose-dependent increase in the revertant number, and the highest value was more than twice the control value with solvent only. Positive controls
2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2) (Ueno Fine Chemical Industries) was used as a positive control without $9 mix for strains TA98 (0.1 /~g/plate) and TA100 (0.01/tg/plate) and 9-aminoacridine (9AA) (Aldrich) for strain TA2637 (100 /~g/plate). With $9 mix, 2-aminoanthracene (2AA) (Aldrich) was used as a positive control for strains TA98 (0.5 #g/plate), TA100 (0.5 /~g/plate) and TA2637 (2 /~g/plate).
Results
6 of the 16 naphthoquinones tested, i.e., plumbagin, naphthazarin, 2-hydroxynaphthoquinone, vitamin K 3 (menadione), juglone and 7-methyljuglone, were mutagenie to strain TA2637 with $9 mix from rat liver. All these compounds, except juglone and 7-methyljuglone, were also slightly mutagenie to strain TA98 with metabolic activation. They all gave negative results with strain TAI00. None of the compounds was mutagenic without metabolic activation. The results of mutagenieity tests on these compounds are presented in Fig. 1. Unsubstituted fl-naphthoquinone, mompain (containing four free hydroxyl groups), all bi-naphthoquinone compounds (i.e., eliptinone, mamegakinone, isodiospyrin, bisisodiospyrin and elsinochrome A), naphthoquinones with a prenyl side chain (i.e., alkannin and vitamin K1) and tanshinone II were non-mutagenie to all the test strains with or without metabolic activation. The results of mutagenicity tests on naphthoquinones are summarized in Table 3, where the specific mutagenic activities (His+/nmole) of the mutagenie compounds are also presented. Plumbagin had the highest specific activity (2.65 revertants/nmole in strain TA2637). Naphthazarin had a specific activity of 1.81, and 2-hydroxynaph-
30
PLUMBAGIN
NAPHTHAZARIN
MENADIONE
800 TA2637: *S9Mix • -$9 Mix A TA98: 600
+59Mix c -59Mix
Q
¢:
>,,
z,O0
o
..= z 200
i
1
10
100
0
20
60
100
140
#g/plate
JUGLONE
2-HYDROXYNAPHTHO-
7-METHYLJUGLONE
OUINONE
TA2637: +S9Mix • -S9Mix * TA98: +S9Mix o -S9Mix
30(
5O0 20C
10C
lOO o
2b ' ' ~ ' " i ~
~
0
10
30 ,ug/plate
5()
0
10
30
5C)
Fig. 1. Results of mutagenicity tests on plumbagin, naphthazarin, vitamin K 3 (menadione), 2-hydroxynaphthoquinone, juglone and 7-methyljuglone. Symbols: TA2637 with (e) and without (*) $9 mix; TA98 with (O) and without (zx) $9 mix. The same symbols are used in all graphs.
thoquinone, vitamin K 3 (menadione), juglone and 7-methyljuglone had values of between about 0.8 and 0.3 His+/nmole. With strain TA98 the specific activity ranged from about 0.3 to 0.1 revertants/nmole. None of the 13 benzoquinones tested was mutagenic in the test system with any of the strains with or without metabolic activation. Compounds were usually tested at 1-100/~g/plate, because of the scarcity of test
31 TABLE 3 MUTAGENICITIES OF NAPHTHOQUINONES TESTED Test compound
Plumbagin Naphthazarin 2-Hydroxynaphthoquinone Vitamin K 3 (menadione) Juglone 7-Methyljuglone fl-Naphthoquinone Mompain Eliptinone Mamegakinone Isodiospyrin Bisisodiospyrin Elsinochrome A Alkannin Tanshinone II Vitamin K1
Mutagenicity TA98 + $9 mix
TAI00 + $9 mix
TA2637 + $9 mix
Specific mutagenic activity (His +/nmole) TA98 TA2637
+ +
-
+ +
0.29 0.37
2.65 1.81
+ + -
-
+ + + + -
0.10 0.07 0 0 0 0 0 0 0 0 0 0 0 0
0.49 0.43 0.84 0.36 0 0 0 0 0 0 0 0 0 0
Symbols: ( - ) not mutagenic, ( + ) revertant number about twice the solvent-control value, ( + ) revertant number more than twice the solvent-control value.
s a m p l e s i s o l a t e d f r o m n a t u r a l sources. T h e t o x i c i t y o f s o m e of t h e c o m p o u n d s also p r e v e n t e d t h e use o f h i g h e r c o n c e n t r a t i o n s . I n the c a s e of r a p a n o n e m o n o m e t h y l e t h e r t h e r e w a s a slight d o s e - d e p e n d e n t i n c r e a s e in t h e r e v e r t a n t n u m b e r in s t r a i n T A 2 6 3 7 w i t h $9 m i x , b u t t h e l e t h a l effect o f the test c o m p o u n d p r e v e n t e d its p o s s i b l e m u t a g e n i c e f f e c t f r o m b e i n g d e t e c t e d b y the p r e - i n c u b a t i o n m e t h o d . D i m e t h y l s u l f o x i d e ( D M S O ) w a s u s e d as a s o l v e n t for all test c o m p o u n d s . D u r i n g t h e c o u r s e o f this w o r k it b e c a m e o b v i o u s t h a t t h e m u t a g e n i c i t y o f s t o c k s o l u t i o n s in D M S O o f all t h e n a p h t h o q u i n o n e s e x c e p t v i t a m i n K 3 d e c r e a s e d d u r i n g s t o r a g e in t h e f r e e z e r ( - 2 0 ° C ) . 2 - H y d r o x y n a p h t h o q u i n o n e was the m o s t stable, its m u t a g e n i c ity d e c r e a s i n g o n l y a b o u t 20% ( c a l c u l a t e d f r o m t h e s p e c i f i c activity) d u r i n g s t o r a g e f o r 1 m o n t h . P l u m b a g i n a n d n a p h t h a z a r i n w e r e less stable, a n d o n s t o r a g e their m u t a g e n i c i t i e s d e c r e a s e d a b o u t 50% in 3 days, a n d a b o u t 90% in 1 m o n t h . 7 - M e t h y l j u g l o n e a n d j u g l o n e w e r e t h e least s t a b l e o n s t o r a g e in D M S O s o l u t i o n : o n l y a fresh s o l u t i o n o f 7 - m e t h y l j u g l o n e was m u t a g e n i c , a n d the m u t a g e n i c i t y o f a s o l u t i o n of j u g l o n e d e c r e a s e d a b o u t 30% in 2 d a y s a n d b e c a m e z e r o a f t e r 10 days. T h e s e results a r e p r e s e n t e d in T a b l e 4. T h e s t a b i l i t y o f v i t a m i n K 3 s o l u t i o n a f t e r s t o r a g e for 40 d a y s was c o n f i r m e d b y t h i n - l a y e r c h r o m a t o g r a p h y . B e c a u s e o f t h e s m a l l a m o u n t s of test m a t e r i a l s a v a i l a b l e , it w a s n o t p o s s i b l e to s t u d y the stabilities o f all n a p h t h o q u i n o n e s in d i f f e r e n t solvents. B u t w e e x a m i n e d
32 TABLE 4 D E C R E A S E 1N S P E C I F I C M U T A G E N I C ACTIVITY OF N A P H T H O Q U I N O N E S S O L U T I O N IN DMSO D U R I N G S T O R A G E IN T H E F R E E Z E R
Age of stock solution
(His +/ nmol e )
(days)
TA98
TA2637
Plumbagin
0 3 30
0.29 0.16
2.65 1.41 0.20
Naphthazarin
0 3 30
0.37 0.23 -
1.81 0.86 0.17
2-Hydroxynaphthoquinone
0 15 30
0.10 0.06
0.49 0.40 0.38
Juglone
0 2 10
0 0 -
0.84 0.60 0
0
0
0.36
1
0
0
Test
compounds
7-Methyljuglone
IN STOCK
Specific mutagenic activity
stock solutions of juglone of different ages by thin-layer chromatography. Results showed that juglone was already partially degraded after storage for 1 day in DMSO solution and that after 10 days no juglone remained in the stock solution. In contrast, in ethan01 juglone was not degraded appreciably after 10 days' storage in the freezer.
Discussion The present results show that naphthoquinones with one or two hydroxyl and/or methyl substituents are mutagenic with metabolic activation. The strongest mutagens were plumbagin and naphthazarin, both having two substituents: plumbagin has one hydroxyl and one methyl group and naphthazarin has two hydroxyl groups. 2-Hydroxynaphthoquinone, juglone and vitamin K 3 all have one substituent - the first two each have a hydroxyl group and vitamin K 3 has a methyl group - and they all have clearly lower mutagenic activities than plumbagin and naphthazarin. It is interesting that, of the mutagenic naphthoquinones tested, 7-methyljuglone had the weakest mutagenic effect, although it has the same substituents as the strongest mutagen, plumbagin: both have a hydroxyl group in position 5, but plumbagin has a methyl group on carbon 2 while 7-methyljuglone has one on carbon 7. Thus it seems that not only the number of substituents, but also the position of substituents is important for mutagenicity.
33 Mompain has four hydroxyl groups in its molecule. We found that mompain is not mutagenic, and it may be that this number of substituents is too large for the compound to be mutagenic. We noticed in previous experiments with anthraquinones (Tikkanen et al., 1983) that the compound with the largest number of simple substituents was the weakest mutagen. Unsubstituted fl-naphthoquinone, naphthoquinones having a prenyl side chain (i.e. alkannin and vitamin Ki) and all bi-naphthoquinone compounds were nonmutagenic in the test system used. Alkannin derivatives are known to have antitumor activity against ascites cells (Sankawa et al., 1981), but they did not have mutagenic activity in this Salmonella test. None of the benzoquinones tested was mutagenic. In addition to hydroxyl and methyl substituents, the benzoquinone compounds tested contained one or more aliphatic side chains; i.e., in this study none of the natural benzoquinones tested had only simple hydroxyl and methyl groups. Hence, the benzoquinones tested differed from the naphthoquinones tested in the number of their benzene rings and in the nature of their substitutions~ Loveless (1951) reported that benzoquinone (apparently unsubstituted) does not have mutagenic (radiomimetic) activity in Vicia. The terphenyl derivatives of benzoquinones that we tested are toxic mold metabolites with cytotoxic effects on HeLa cells (Takahashi et al., 1976), but according to our results they were not mutagenic. According to our study and an earlier report (Tikkanen et al., 1983), natural quinones are mutagenic if they have three (anthraquinones) or two (naphthoquinones) hydrocarbon rings with simple hydroxyl and/or methyl substituents, anthraquinones being the strongest mutagens. Their mutagenicity'seems to decrease when the number of hydroxyl substituents becomes large, i.e. four hydroxyl groups per molecule. Quinone pigments are widely distributed in nature and are extensively used by man. Thus it is important to clarify their toxicological proporties, especially their long-term effects, by investigating their mutagenicity and also possible carcinogenicity.
Acknowledgements
We thank Dr. U. Sankawa, Faculty of Pharmaceutical Sciences, University of Tokyo, Tokyo, for supplying alkannin and Dr. H. Kakisawa, Department of Chemistry, Tsukuba University, Ibaraki, for supplying tanshinone II. This study was supported in part by a Grant-in-Aid for Research on Environmental Science from the Ministry of Education, Science and Culture of Japan, and by a grant from the U.S.-Japan Cooperative Medical Science Program.
References Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test, Mutation Res., 31,347-364.
34 Chen, C.-T., K. Nakanishi and S. Natori (1966) Biosynthesis of elsinochrome A, the perylenequinone from Elsinoe spp. I, Chem. Pharm. Bull., 14, 1434-1437. Ikekawa, T., E.L. Wang, M. Hamada, T. Takeuchi and H. Umezawa (1967) Isolation and identification of the antifungal active substance in walnuts, Chem. Pharm. Bull., 15, 242-245. Loveless, A. (1951) Qualitative aspects of the chemistry and biology of radiomimetic (mutagenic) substances, Nature (London), 167, 338-342. Matsushima, T., M. Sawamura, K. Hara and T. Sugimura (1976) A safe substitute for polychlorinated biphenyls as an inducer of metabolic activation system, in: F.J. de Serres, J.R. Fouts, J.R. Bend and R.M. Philpot (Eds.), In Vitro Metabolic Activation in Mutagenesis Testing, Elsevier/North-Holland, Amsterdam, pp. 85-88. Natori, S., H. Nishikawa and H. Ogawa (1964) Structure of helicobasidin, a novel benzoquinone from Helicobasidium mompa Tanaka, Chem. Pharm. Bull., 12, 236-243. Natori, S., Y. Inouye and H. Nishikawa (1967) The structures of mompain and deoxyhelicobasidin and the biosynthesis of helicobasidin, quinonoid metabolites of Helicobasidiurn mompa Tanaka, Chem. Pharm. Bull., 15, 380-390. Ogawa, H., and S. Natori (1965) Hydroxybenzoquinones from Myrsinaceae plants, I. Reconfirmation of the structure of maesaquinone and isolation of acetylmaesaquinone from Maesa Japonica Moritzi, Chem. Pharm. Bull., 13, 511-516. Ogawa, H., and S. Natori (1968a) Hydroxybenzoquinones from Myrsinaceae plants, II. Distribution among Myrsinaceae plants in Japan, Phytochemistry, 7, 773-782. Ogawa, H., and S. Natori (1968b) Hydroxybenzoquinones from Myrsinaceae plants, III. The structures of 2-hydroxy-5-methoxy-3-pentadecenylbenzoquinone and ardisiaquinones A, B and C from Ardisia spp., Chem. Pharm. Bull., 16, 1709-1720. Okumura, Y., H. Kakisawa, M. Kato and Y. Hirata (1961) The structure of tanshinone-lI, Bull. Chem. Soc. Japan, 34, 895-897. Sankawa, U., H. Otsuka, Y. Kataoka, Y. Iitaka, A. Hoshi and K. Kuretani (1981) Antitumor activity of shikonin, alkannin and their derivatives, II. X-ray analysis of cyclo-alkannin leucoacetate, tautomerism of alkannin and cyclo-alkannin and antitumor activity of alkannin derivatives, Chem. Pharm. Bull., 29, 116-122. Takahashi, C., K. Yoshihira, S. Natori and M. Umeda (1976) The structures of toxic metabolites of Aspergillus candidus, I. The compounds A and E, cytotoxic p-terphenyls, Chem. Pharm. Bull., 24, 613-620. Tezuka, M., M. Kuroyanagi, K. Yoshihira and S. Natori (1972) Naphthoquinone derivatives from the Ebenaceae, IV. Naphthoquinone derivatives from Diospyros kaki Thunb. and D. kaki Thunb. vat sylvestris Makino, Chem. Pharm. Bull., 20, 2029-2035. Tezuka, M., C. Takahashi, M. Kuroyanagi, M. Satake, K. Yoshihira and S. Natori (1973) New naphthoquinones from Diospyros, Phytochemistry, 12, 175-183. Thomson, R.H. (1971) Naturally occurring quinones, Academic Press, London. Tikkanen, L., T. Matsushima and S. Natori (1983) Mutagenicity of anthraquinones in the Salmonella preincubation test, Mutation Res., 116, 297-304. Yahagi, T., M. Degawa, Y. Seino, T. Matsushima, M. Nagao, T. Sugimura and Y. Hashimoto (1975) Mutagenicity of carcinogenic azo dyes and their derivatives, Cancer Lett., 1, 91-96. Yamaguchi, K. (1942) Synthesis of DL-dihydroperezone, J. Pharm. Soc. Japan, 62, 491-499 (in Japanese). Yoshihira, K., M. Tezuka and S. Natori (1971) Naphthoquinone derivatives from the Ebenaceae, II. Isodiospyrin, bisisodiospyrin, and mamegakinone from Diospyros lotus L. and D. morrisiana Hance, Chem. Pharm. Bull., 19, 2308-2313.
25
Elsevier MTR 00805
Mutagenicity of natural n a p h t h o q u i n o n e s and b e n z o q u i n o n e s in the S a l m o n e l l a / m i c r o s o m e test Leena Tikkanen 1,,, Taijiro Matsushima 1,**, Shinsaku Natori 2,*** and Kunitoshi Yoshihira 2 l Department of Molecular Oncology, Institute of Medical Science, Universityof Tokyo, Shirokanedai, Minato- ku, Tokyo 108; and 2 National Institute of Hygienic Sciences, Kamiyoga, Setagaya. ku, Tokyo 158 (Japan)
(Received2 March 1983) (Accepted 11 May 1983)
Summary The mutagenicities of naturally occurring naphthoquinones and benzoquinones were tested by the pre-incubation method with Salmonella typhimurium strains TA98, TA100 and TA2637, which all contain plasmid pKM101. 6 of the 16 naphthoquinones tested, i.e., plumbagin, naphthazarin, 2-hydroxynaphthoquinone, vitamin K 3 (menadione), juglone and 7-methyljuglone, were mutagenic to strain TA2637 with metabolic activation. Except for juglone and 7-methyljuglone, these compounds also had slight mutagenic effects on strain TA98 with $9 mix. All the mutagenic naphthoquinones contain one or two hydroxyl a n d / o r methyl substituents. The naphthoquinone mompain, which has four hydroxyl groups, was not mutagenic. Unsubstituted fl-naphthoquinone, naphthoquinones with a prenyl side chain and all bi-naphthoquinone derivatives tested were non-mutagenic. None of the 13 benzoquinones examined was mutagenic to any of the strains used with or without metabolic activation. These results show that natural naphthoquinones are mutagenic when they have only one or two hydroxyl a n d / o r methyl substituents.
Quinone pigments are a large class of naturally occurring coloring substances present in various families of plants and fungi. The largest group of naturally * Recipient of a scholarship from the Japanese government during the period of this work. Present address: TechnicalResearch Centre of Finland, Food Research Laboratory,Biologinkuja1, SF-02150 Espoo i 5, Finland. ** To whom requests for reprints should be addressed. *** Present address: Meiji Collegeof Pharmacy,Yato-cho,Tanashi-shi, Tokyo 188 (Japan). 0165-1218/83/$03.00 © 1983 ElsevierSciencePublishers B.V.
26 occurring quinones is that of anthraquinones, but naphthoquinones and benzoquinones are also common. Most naphthoquinones are plant products, whereas benzoquinones have been found in higher plants and in fungi (Thomson, 1971). Substituted 1,4-naphthoquinones occur in nature as various pigments and as K vitamins. Several of these compounds, such as lawsone (2-hydroxynaphthoquinone), alkannin and its antipode (shikonin), are used as dyestuffs. Juglone, methyljuglone and plumbagin are widely used in folk medicines (Thomson, 1971). K vitamins are widely distributed in higher green plants; they are used as prothrombogenic vitamins. Some hydroxybenzoquinones are widely distributed in Myrsinaceae plants (Thomson, 1971; Ogawa and Natori, 1968a, 1968b). Some of these benzoquinones are used for medical purposes because they have anthelminthic and purgative properties. Terphenyl derivatives of benzoquinones occur mainly in fungi as colored and toxic components (Thomson, 1971). Ubiquinones are a group of benzoquinones involved in electron transport in mitochondrial preparations. They are present in most aerobic organisms from bacteria to higher plants and animals. Although naphthoquinones and benzoquinones are widely distributed in nature and are extensively used by man for various purposes, there have been few reports about their toxicological properties, including their mutagenicities and carcinogenicities. In the present study, we examined the mutagenicities of natural naphthoquinones and benzoquinones by the pre-incubation method with Salmonella typhimurium strains TA98, TA100 and TA2637.
Materials and methods
Test compounds The naphthoquinones and benzoquinones tested, and their structures, sources and references to the methods used for their isolation are presented in Tables 1 and 2. The naphthoquinones naphthazarin, 2-hydroxynaphthoquinone,/3-naphthoquinone, vitamin K 3 (menadione) and vitamin K~ were purchased from Tokyo Kasei Kogyo Co., Tokyo. Plumbagin, juglone, 7-methyljuglone, mompain, eliptinone, mamegakinone, isodiospyrin, bisisodiospyrin and elsinochrome A were isolated at the National Institute of Hygienic Sciences, Tokyo. Alkannin was a generous gift from Dr. U. Sankawa, Faculty of Pharmaceutical Sciences, University of Tokyo, Tokyo. Tanshinone II was a generous gift from Dr. H. Kakisawa, Department of Chemistry, Tsukuba University, Ibaraki. The benzoquinone ubiquinone-9 was purchased from Taisho Pharmaceutical Co., Tokyo. Embelin, rapanone, maesaquinone, dihydromaesaquinone, rapanone monomethyl ether, rapanone dimethyl ether, helicobasidin, (+)-dihydroperezone, ardisiaquinone A, ardisiaquinone B, 2,5-di-p-anisyl-p-benzoquinone and 2,5-di-(pacetoxyphenyl)-3-acetoxy-p-benzoquinonewere isolated or prepared at the National Institute of Hygienic Sciences, Tokyo. The purities of the test compounds were confirmed by physical data and by thin-layer chromatography.
27
TABLE 1 STRUCTURES AND SOURCES OF NAPHTHOQUINONES TESTED NAME
STRUCTURE
SOURCE
REFERENCE (Isolation method)
o 1.
PLUMBAGIN
~M* No o
DlOspyros kaki
2.
NAPHTNAZARIN
~
3.
2-HYDROXYNAPHTHO- ~ o N QUINONE o
synthetic
4.
MENADIONE
@..
synthetic
5.
JUGLONE
oH~o
Juglsns regis
Ikekews, et el., 1967
6.
7-METHYLJUGLONE M e ~
Dlospyros lotus
Yoshihlra, et el., 1971 Tezuka, et el., 1973
Hollcobusldlum mompa
Netorl, et el.,
Tezuks, et el., 1972, 1973
oH o
synthetic o
o oH o oH o 7.
MOMPAIN
8.
fl-NAPHTHOQUINONE
9.
ELIPTINONE
~ Ho~OH o
cr;r ° o oH
10.
MAMEGAKINONE
11.
ISOOIOSPYRIN
12.
BISISOOIOSPYRIN
13.
ELSINOCHROME A
1967
synthetic oH o
Dlospyros maritime Me
Tezuke, et el., 1973
Me
OH o 0014 u * ~ ~ o'~ o ''~'Me Dloepyros lotus oH o ~ Me~o Me oM o
HO
Dloepyros lotus
OH 0
O HOe
OH
Yoshlhlra, et el., 1971 Tezuka, et el., 1973 Yoshlhlre, et el., 1971 Tezuks, et el., 1973
Yoshihire, et el., 1971 Tezuke, et el., 1973
Dloepyros lotus o[k O0uH 0M* ~ MOO COMI MoO~ O
Elslnoee erellee
Chen, et el., 1966
Mecrotomle euchrome
Senkawe, et el.0 1981
Salvia mlltlorrhlzs
Okumure, et el., 1961
O~ M COMe
O,H (~MeMe Me~U~OH
14. ALKANNIN
16.
TANSHINONE II
19.
VITAMIN K 1
~
@'" °7
°.:cH-(~ °.=°.:°.2~:.
c.:
synthetic
28 TABLE 2 S T R U C T U R E S A N D SOURCES OF B E N Z O Q U I N O N E S TESTED NAME
STRUCTURE
SOURCE
o•c
H
1. EMBELIN
H
11H23
HO
C13H27
2. RAPANONE
REFERENCE ( Isolation method)
Myrslne sequlnll
Ogawa and Natorl, 1968 a
Ardlsla crenata
Ogawe and Natorl, 1 9 6 8 a
Masse Japonica
Ogawa and Natorl, 1 9 6 5
partial synthesis
Ogawa and Natorl, 1 9 6 5
partial synthesis
O g a w l end Hatori, 1 9 6 8 b
partial synthesis
Ogawa and Netorl, 1 9 6 8 b
O 3. MAESAOUINONE H
H2113CH:CH-C4H 9
0 Me~)H
4. DIHYDROMAESAQUINONE
HO
5. RAPANONE MONOMETHYL ETHER
C19H39
Meo
c13~27 o
6. RAPANONE DIMETHYL ETHER
MeO
7. UBIQUINONE-9
O CIH3 MeO~U~(C H2CHIC-CH2)SH MIO" Y "M•
13H27
synthetic
0
""oH_
8. HELICOBASIDIN
H
9. (::~)-D|H YORO PEREZONE
Hellcobasldlum momba
Natorl, et el., 1 9 6 4
synthetic
Yamaguchi, 1942
Ardisia sleboldll
Ogawa and Natorl, 1 9 6 8 b
Ardlsla sleboldli
Ogawa end Nalorl, 1 9 6 8 b
~--- Oue
synthetic
Takahaahi, i t el., 1 9 7 6
OAc
synthetic
Tekahashl, et el., 1 9 7 6
Me Ma Me O Me
~ Me
10, ARDISIAOUINONE A
~l~ 10H
HO- y
(CH)
H
11. AROISIAOUINONE B
0
12. 2 , 5 - D I - p - A N I S Y L - p BENZOOUINONE
MeO.~--~
13. 2,5-DI-(p-ACETOXYPHENYL)-3-ACETOXY-pBENZOOUINONE
,
o 4
c
0
~
Bacterial strains Salmonella typhimurium strains TA100, TA2637 and TA98 were used as tester strains. These strains were derived from S. typhimurium strains TA1535, TA1537 and TA1538, respectively, by introduction of plasmid pKM101 (Ames et al., 1975). The tester strains were provided by Dr. B.N. Ames, University of California, Berkeley, California. They were grown overnight in nutrient broth (Difco) with shaking before use.
29 Mutation test
The Salmonella mutation test was performed by the pre-incubation method, a modification of the plate-test method (Yahagi et al., 1975). A mixture of the test compound in 50/~1 of dimethylsulfoxide, 0.5 ml of $9 mix or 0.2 M phosphate buffer (pH 7.4) and 100/~1 of culture of the tester strain was pre-incubated for 30 min at 30°C in a test-tube. Then 2 ml of top agar containing 0.5 mM histidine and biotin was added, and after thorough mixing, the solution was poured on to Vogel-Bonner minimal agar plates. The plates were incubated for 48 h at 37°C, and then revertant colonies were counted. Liver homogenate ($9) was prepared by the method of Ames et al. (1975) from the livers of rats treated with Na-phenobarbital and 5,6-benzoflavone (Matsushima et al., 1976). $9 mix contained (per ml) 100/~1 of $9, 4/tmoles each of NADPH and NADH, 5 /tmoles of glucose-6-phosphate, 33 /~moles of KC1, 8 ~tmoles of MgC12 and 100/~moles of Na-phosphate buffer, pH 7.4. The result was defined as positive when the compound induced a dose-dependent increase in the revertant number, and the highest value was more than twice the control value with solvent only. Positive controls
2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2) (Ueno Fine Chemical Industries) was used as a positive control without $9 mix for strains TA98 (0.1 /~g/plate) and TA100 (0.01/tg/plate) and 9-aminoacridine (9AA) (Aldrich) for strain TA2637 (100 /~g/plate). With $9 mix, 2-aminoanthracene (2AA) (Aldrich) was used as a positive control for strains TA98 (0.5 #g/plate), TA100 (0.5 /~g/plate) and TA2637 (2 /~g/plate).
Results
6 of the 16 naphthoquinones tested, i.e., plumbagin, naphthazarin, 2-hydroxynaphthoquinone, vitamin K 3 (menadione), juglone and 7-methyljuglone, were mutagenie to strain TA2637 with $9 mix from rat liver. All these compounds, except juglone and 7-methyljuglone, were also slightly mutagenie to strain TA98 with metabolic activation. They all gave negative results with strain TAI00. None of the compounds was mutagenic without metabolic activation. The results of mutagenieity tests on these compounds are presented in Fig. 1. Unsubstituted fl-naphthoquinone, mompain (containing four free hydroxyl groups), all bi-naphthoquinone compounds (i.e., eliptinone, mamegakinone, isodiospyrin, bisisodiospyrin and elsinochrome A), naphthoquinones with a prenyl side chain (i.e., alkannin and vitamin K1) and tanshinone II were non-mutagenie to all the test strains with or without metabolic activation. The results of mutagenicity tests on naphthoquinones are summarized in Table 3, where the specific mutagenic activities (His+/nmole) of the mutagenie compounds are also presented. Plumbagin had the highest specific activity (2.65 revertants/nmole in strain TA2637). Naphthazarin had a specific activity of 1.81, and 2-hydroxynaph-
30
PLUMBAGIN
NAPHTHAZARIN
MENADIONE
800 TA2637: *S9Mix • -$9 Mix A TA98: 600
+59Mix c -59Mix
Q
¢:
>,,
z,O0
o
..= z 200
i
1
10
100
0
20
60
100
140
#g/plate
JUGLONE
2-HYDROXYNAPHTHO-
7-METHYLJUGLONE
OUINONE
TA2637: +S9Mix • -S9Mix * TA98: +S9Mix o -S9Mix
30(
5O0 20C
10C
lOO o
2b ' ' ~ ' " i ~
~
0
10
30 ,ug/plate
5()
0
10
30
5C)
Fig. 1. Results of mutagenicity tests on plumbagin, naphthazarin, vitamin K 3 (menadione), 2-hydroxynaphthoquinone, juglone and 7-methyljuglone. Symbols: TA2637 with (e) and without (*) $9 mix; TA98 with (O) and without (zx) $9 mix. The same symbols are used in all graphs.
thoquinone, vitamin K 3 (menadione), juglone and 7-methyljuglone had values of between about 0.8 and 0.3 His+/nmole. With strain TA98 the specific activity ranged from about 0.3 to 0.1 revertants/nmole. None of the 13 benzoquinones tested was mutagenic in the test system with any of the strains with or without metabolic activation. Compounds were usually tested at 1-100/~g/plate, because of the scarcity of test
31 TABLE 3 MUTAGENICITIES OF NAPHTHOQUINONES TESTED Test compound
Plumbagin Naphthazarin 2-Hydroxynaphthoquinone Vitamin K 3 (menadione) Juglone 7-Methyljuglone fl-Naphthoquinone Mompain Eliptinone Mamegakinone Isodiospyrin Bisisodiospyrin Elsinochrome A Alkannin Tanshinone II Vitamin K1
Mutagenicity TA98 + $9 mix
TAI00 + $9 mix
TA2637 + $9 mix
Specific mutagenic activity (His +/nmole) TA98 TA2637
+ +
-
+ +
0.29 0.37
2.65 1.81
+ + -
-
+ + + + -
0.10 0.07 0 0 0 0 0 0 0 0 0 0 0 0
0.49 0.43 0.84 0.36 0 0 0 0 0 0 0 0 0 0
Symbols: ( - ) not mutagenic, ( + ) revertant number about twice the solvent-control value, ( + ) revertant number more than twice the solvent-control value.
s a m p l e s i s o l a t e d f r o m n a t u r a l sources. T h e t o x i c i t y o f s o m e of t h e c o m p o u n d s also p r e v e n t e d t h e use o f h i g h e r c o n c e n t r a t i o n s . I n the c a s e of r a p a n o n e m o n o m e t h y l e t h e r t h e r e w a s a slight d o s e - d e p e n d e n t i n c r e a s e in t h e r e v e r t a n t n u m b e r in s t r a i n T A 2 6 3 7 w i t h $9 m i x , b u t t h e l e t h a l effect o f the test c o m p o u n d p r e v e n t e d its p o s s i b l e m u t a g e n i c e f f e c t f r o m b e i n g d e t e c t e d b y the p r e - i n c u b a t i o n m e t h o d . D i m e t h y l s u l f o x i d e ( D M S O ) w a s u s e d as a s o l v e n t for all test c o m p o u n d s . D u r i n g t h e c o u r s e o f this w o r k it b e c a m e o b v i o u s t h a t t h e m u t a g e n i c i t y o f s t o c k s o l u t i o n s in D M S O o f all t h e n a p h t h o q u i n o n e s e x c e p t v i t a m i n K 3 d e c r e a s e d d u r i n g s t o r a g e in t h e f r e e z e r ( - 2 0 ° C ) . 2 - H y d r o x y n a p h t h o q u i n o n e was the m o s t stable, its m u t a g e n i c ity d e c r e a s i n g o n l y a b o u t 20% ( c a l c u l a t e d f r o m t h e s p e c i f i c activity) d u r i n g s t o r a g e f o r 1 m o n t h . P l u m b a g i n a n d n a p h t h a z a r i n w e r e less stable, a n d o n s t o r a g e their m u t a g e n i c i t i e s d e c r e a s e d a b o u t 50% in 3 days, a n d a b o u t 90% in 1 m o n t h . 7 - M e t h y l j u g l o n e a n d j u g l o n e w e r e t h e least s t a b l e o n s t o r a g e in D M S O s o l u t i o n : o n l y a fresh s o l u t i o n o f 7 - m e t h y l j u g l o n e was m u t a g e n i c , a n d the m u t a g e n i c i t y o f a s o l u t i o n of j u g l o n e d e c r e a s e d a b o u t 30% in 2 d a y s a n d b e c a m e z e r o a f t e r 10 days. T h e s e results a r e p r e s e n t e d in T a b l e 4. T h e s t a b i l i t y o f v i t a m i n K 3 s o l u t i o n a f t e r s t o r a g e for 40 d a y s was c o n f i r m e d b y t h i n - l a y e r c h r o m a t o g r a p h y . B e c a u s e o f t h e s m a l l a m o u n t s of test m a t e r i a l s a v a i l a b l e , it w a s n o t p o s s i b l e to s t u d y the stabilities o f all n a p h t h o q u i n o n e s in d i f f e r e n t solvents. B u t w e e x a m i n e d
32 TABLE 4 D E C R E A S E 1N S P E C I F I C M U T A G E N I C ACTIVITY OF N A P H T H O Q U I N O N E S S O L U T I O N IN DMSO D U R I N G S T O R A G E IN T H E F R E E Z E R
Age of stock solution
(His +/ nmol e )
(days)
TA98
TA2637
Plumbagin
0 3 30
0.29 0.16
2.65 1.41 0.20
Naphthazarin
0 3 30
0.37 0.23 -
1.81 0.86 0.17
2-Hydroxynaphthoquinone
0 15 30
0.10 0.06
0.49 0.40 0.38
Juglone
0 2 10
0 0 -
0.84 0.60 0
0
0
0.36
1
0
0
Test
compounds
7-Methyljuglone
IN STOCK
Specific mutagenic activity
stock solutions of juglone of different ages by thin-layer chromatography. Results showed that juglone was already partially degraded after storage for 1 day in DMSO solution and that after 10 days no juglone remained in the stock solution. In contrast, in ethan01 juglone was not degraded appreciably after 10 days' storage in the freezer.
Discussion The present results show that naphthoquinones with one or two hydroxyl and/or methyl substituents are mutagenic with metabolic activation. The strongest mutagens were plumbagin and naphthazarin, both having two substituents: plumbagin has one hydroxyl and one methyl group and naphthazarin has two hydroxyl groups. 2-Hydroxynaphthoquinone, juglone and vitamin K 3 all have one substituent - the first two each have a hydroxyl group and vitamin K 3 has a methyl group - and they all have clearly lower mutagenic activities than plumbagin and naphthazarin. It is interesting that, of the mutagenic naphthoquinones tested, 7-methyljuglone had the weakest mutagenic effect, although it has the same substituents as the strongest mutagen, plumbagin: both have a hydroxyl group in position 5, but plumbagin has a methyl group on carbon 2 while 7-methyljuglone has one on carbon 7. Thus it seems that not only the number of substituents, but also the position of substituents is important for mutagenicity.
33 Mompain has four hydroxyl groups in its molecule. We found that mompain is not mutagenic, and it may be that this number of substituents is too large for the compound to be mutagenic. We noticed in previous experiments with anthraquinones (Tikkanen et al., 1983) that the compound with the largest number of simple substituents was the weakest mutagen. Unsubstituted fl-naphthoquinone, naphthoquinones having a prenyl side chain (i.e. alkannin and vitamin Ki) and all bi-naphthoquinone compounds were nonmutagenic in the test system used. Alkannin derivatives are known to have antitumor activity against ascites cells (Sankawa et al., 1981), but they did not have mutagenic activity in this Salmonella test. None of the benzoquinones tested was mutagenic. In addition to hydroxyl and methyl substituents, the benzoquinone compounds tested contained one or more aliphatic side chains; i.e., in this study none of the natural benzoquinones tested had only simple hydroxyl and methyl groups. Hence, the benzoquinones tested differed from the naphthoquinones tested in the number of their benzene rings and in the nature of their substitutions~ Loveless (1951) reported that benzoquinone (apparently unsubstituted) does not have mutagenic (radiomimetic) activity in Vicia. The terphenyl derivatives of benzoquinones that we tested are toxic mold metabolites with cytotoxic effects on HeLa cells (Takahashi et al., 1976), but according to our results they were not mutagenic. According to our study and an earlier report (Tikkanen et al., 1983), natural quinones are mutagenic if they have three (anthraquinones) or two (naphthoquinones) hydrocarbon rings with simple hydroxyl and/or methyl substituents, anthraquinones being the strongest mutagens. Their mutagenicity'seems to decrease when the number of hydroxyl substituents becomes large, i.e. four hydroxyl groups per molecule. Quinone pigments are widely distributed in nature and are extensively used by man. Thus it is important to clarify their toxicological proporties, especially their long-term effects, by investigating their mutagenicity and also possible carcinogenicity.
Acknowledgements
We thank Dr. U. Sankawa, Faculty of Pharmaceutical Sciences, University of Tokyo, Tokyo, for supplying alkannin and Dr. H. Kakisawa, Department of Chemistry, Tsukuba University, Ibaraki, for supplying tanshinone II. This study was supported in part by a Grant-in-Aid for Research on Environmental Science from the Ministry of Education, Science and Culture of Japan, and by a grant from the U.S.-Japan Cooperative Medical Science Program.
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