Anticlastogenic effect of flavonoids against mutagen-induced micronuclei in mice

Mutation Research, 284 (1992)243-249

243

© 1992ElsevierSciencePublishers B.V. All rights reserved0027-5107/92/$05.00 MUT 05182

Anticlastogenic effect of flavonoids against mutagen-induced micronuclei in mice M.Y. H e o a, K.S. Yu a K.H. Kim a, H.P. Kim a and W.W. Au b " College of Pharmacy, Kangweon National Unicersity, Chuncheon 200-701, South Korea and h Department of Precentice Medicine and Community Health, Unicersity of Texas Medical Branch, Gah'eston, TX 77550, USA

(Received9 January 1992) (Revision received26 June 1992) (Accepted 30 June 1992)

Keywords: Flavonoid; Anticlastogenic effect; Micronucleus test; Benzo[a]pyrene;Galangin; Ethyl methanesulphonate; 7,12-Di-

methylbenz[a]anthracene;Adriamycin

Summary 14 flavonoids, including flavone and flavonol derivatives, were tested for their anticlastogenic effect against induction of micronuclei by benzo[a]pyrene in polychromatic erythrocytes of mice. When each flavonoid was administered orally, together with intraperitoneally administered benzo[a]pyrene, most flavonol derivatives showed an anticlastogenic effect. The data suggest that the 2,3-double bond and 3,5,7-hydroxyl groups in the flavonoid molecules may be essential to produce anticlastogenic effects against benzo[a]pyrene. Galangin, one of the active compounds, and (-)-epicatechin, a weak one, were administered to mice in order to compare their anticlastogenic effect against 3 different kinds of carcinogens: ethyl methanesulfonate, 7,12-dimethylbenz[a]anthracene, and adriamycin. Galangin showed a stronger anticlastogenic effect than (-)-epicatechin against ethyl methanesulfonate and 7,12-dimethylbenz[a]anthracene. However, there was no significant effect against adriamycin-induced micronuclei by both compounds. Our study indicates that most flavonoids are anticlastogenic agents. Their anticlastogenic effects are apparently independent of their own clastogenic activities. Furthermore, their anticlastogenic activities do not apply universally to all types of genotoxic chemicals.

In recent years attention has been focused on whether naturally occurring compounds can modify the mutagenic and carcinogenic effects of environmental contaminants. Among these natural compounds are flavonoids, and they are widely distributed in the plant kingdom. The average

Correspondence: Dr. H.P. Kim, Ph.D., Associate Professor, College of Pharmacy, Kangweon National University, Chuncheon 200-701, South Korea.

daily intake of all flavonoids by humans is estimated to be about 1 g per day (Herman, 1976; Brown, 1980). Because a large amount of flavonoids are consumed by humans, these compounds have been intensively studied for their genotoxic/carcinogenic potential and for their interaction with mutagenic and carcinogenic compounds (Brown, 1980; Pamukcu et al., 1980; Metlz and MacGregor, 1981; MacGregor, 1986; Wall et al., 1988). Although the mutagenicity of some flavonoids such as quercetin is well established in

244

the prokaryotic system (Nagao et al., 1981; Elliger et al., 1984), the limited genotoxic studies of flavonoids in the eukaryotic system have yielded conflicting results (Carver et al., 1983; Van der Hoeven et al., 1984; Rueff et al., 1986; Popp and Schimmer, 1991). It is therefore generally believed that flavonoids are not potent genotoxic agents in eukaryotes. Some investigators have documented that flavonoids suppress the mutagenicity of various chemicals. Huang et al. (1983) reported that several flavonoids reduced the mutagenic activity of benzo[a]pyrene-7,8-diol-9,10-epoxide in TA100 strain of Salmonella typhimurium. Similar antimutagenic effects against N-methyl-N'-nitro-Nnitrosoguanidine and aflatoxin BI in bacteria were reported by Frances et al. (1989). On the other hand, some flavonoids enhanced mutation by 2acetylaminofluorene in bacteria (Ogawa et al., 1985). Most of these studies have been conducted with two groups of flavonoids. Representative flavonoid compounds are shown in Fig. 1. Other than flavanol and flavonol, the activities of the other groups have not been adequately investigated. In addition, few studies have been conducted with mammalian cells, especially in in vivo systems. Therefore, we have investigated the clastogenic effects of 5 different groups of flavonoids and their anticlastogenic effects against the induction of micronuclei by benzo[a]pyrene in bone-marrow polychromatic erythrocytes of mice.

2

~

3

2

~

3' 6

8

4' 5'

7

6'

6 5

0

~,z.3

3

5

7

2'

3'

4'

5'

no

H

H

H

H

H

H

H H

Flavanone Narlngenin

no

H

OH

OH

H

H

OH

Flavone

yes

H

H

H

H

H

H

H

Flavonol

yes

OH

H

H

H

H

H

H

Chrysin

yes

H

OH

OH

H

H

H

H

Apigenin

yes

H

OH

OH

H

H

OH

H

Galangin

yes

OH

OH

OH

H

H

H

H

Kaempferol

yes

OH

OH

OH

H

H

OH

H

Ouercet[n

yes

OH

OH

OH

H

OH

OH

H

~rin

yes

OH

OH

OH

H

OH

H

t~yrlcetln

yes

OH

OH

OH

OH

OH

OH

OH

~ O H

(-)-Epiemthechin

R = 4 OH

( ~ ) - G a t e , chin

R = ~ OH

OH

H

HOooH3 Biochanin A

Fig. 1. Chemical structure of the flavonoid derivatives, z~e.3 means double bond at the 2 and 3 positions of C-ring.

Materials and methods

kaempferol (520-18-3); morin (480-16-0); myricetin (529-44-2); naringenin (480-41-1); quercetin (651-25-3).

Materials. Flavonol and kaempferol were obtained from Tokyo Kasei Chemical Company, Tokyo, Japan. Other flavonoids were purchased from Aldrich Chemical Company, Milwaukee, Wisconsin, USA (Fig. 1). Benzo[a]pyrene [B(a)P; CAS No. 50-32-8], ethyl methanesulfonate (EMS; CAS No. 62-50-0), 7,12-dimethylbenz[a]anthracene (DMBA; CAS No. 57-97-6), and adriamycin (ADM; CAS No. 25316-40-9) were supplied by Sigma Chemical Company, St. Louis, MO, USA. The CAS Nos. for the various flavonoids are: apigenin (520-36-5); biochanin A (491-80-5); catechin (88191-48-4); chrysin (480-40-0); epicatechin (490-46-0); fisetin (528-48-3); flavanone (487-263); flavone (525-82-6); galangin (548-83-4);

Animal treatment and micronucleus test. Male ICR mice (20-25 g) which were obtained from the Animal Center of the Catholic Medical School (Seoul, Korea) were used throughout the experiments. Animals were maintained in chambers with laminar air flow at a temperature of 22 _+ I°C and relative humidity of 55 +_ 7% throughout the study. Each flavonoid was administered orally once or every day for 5 days to 3 mice per dose and per chemical. The carcinogens (B(a)P, EMS, DMBA, or ADM) were administered intraperitoneally and immediately after the single or last dose of flavonoid. Mice administered corn oil were used as vehicle controls. The treated ani-

245 2.0

1.5

1.0-

0.5-

i

I

[

i

i

i

1

i

[

2

6

12

18

24

30

36

48

72

Time after B(a)P injection (hrs) Fig. 2. Time-dependent increase of micronuclei in polychromatic erythrocytes (MNPCE) in bone-marrow cells of mice treated with benzo[a]pyrene. Benzo[a]pyrene (150 m g / k g ) was intraperitoneally injected. 1000 cells were analyzed to determine micronuclei frequencies. Data points and bars represent mean + SD of 3 mice per group.

mals were sacrificed by cervical dislocation at 36 h after the treatment with carcinogens. Mice treated with flavonoid or corn oil were sacrificed at 36 h after the last treatment. In another experiment mice were treated with different doses and for different times with B(a)P to elucidate a dose-

and time-dependent effect. The bone-marrow preparations were carried out as previously reported by Schmid (1975), and 1000 polychromatic erythrocytes (PCEs) were scored per animal to determine the frequency of micronucleated polychromatic erythrocytes (MNPCEs). Analysis of

2.0"

Y= 0,2433 + 0 . 0 0 8 4 X

~

]

~

( r=0.9854 )

'~ 1.5-

n z 1.0

0.5

0 0

i

J

i

,

i

25

50

100

150

200

Bla)P

dose

,

mg/kg

Fig. 3. Effects of benzo[a]pyrene concentration on MNPCE in mouse bone-marrow cells. Mice were sacrificed after 36 h of benzo[a]pyrene injection. Data points and bars represent mean + SD of 3 mice per group.

246

dose-dependent effect of B(a)P. Fig. 3 shows that a good dose-response induction of MNPCE was caused by treatment of mice with 25-200 m g / k g body weight of B(a)P. Therefore, in the subsequent studies, mice were sacrificed at 36 h after administration of 150 m g / k g B(a)P. 14 flavonoid derivatives were singly administered at 0.1, 1 and 10 m g / k g body weight with a concomitant injection of B(a)P. Among them, galangin, kaempferol, morin, and quercetin showed potent anticlastogenic effects on B(a)P-induced micronucleus in mouse bone-marrow ceils (Table 1). When the flavonoid derivatives were multiply administered for 5 days, galangin, morin and myricetin showed potent anticlastogenic effect (more than 30%

Variance and Students' t-test were used for the statistical analysis. Results

In order to establish the proper treatment time and the dose level of B(a)P, various concentrations of B(a)P were administered intraperitoneally, and mice were sacrificed at different times for the MN analysis. As shown in Fig. 2, the frequencies of MNPCEs in mouse bone-marrow cells show a time-dependent increase (from 0 to 72 h) with a plateau at 36-48 h when 150 m g / k g body weight B(a)P was used. A treatment time of 36 h was therefore chosen to determine the

TABLE 1 S U P P R E S S I O N O F B(a)P-INDUCED M N P C E s BY SINGLE T R E A T M E N T O F F L A V O N O I D S IN B O N E - M A R R O W CELLS OF MICE ~' Flavonoids

M N P C E (% and range) b 0.1 ~

1.0

10.0

1.50 (1.3-1.7) d

1.30 (1.3-1.3)

0.95 (I).9-1.0)

1.77 (1.6-1.9) 1.60 1.6-1.6)

1.67 (I.5-1.9) 1.53 (1.0-1.8)

1.47 (1.3-1.6) 1.67 (1.5 1.8)

1.60 1.5-1.7) 1.67 1.4-1.9)

1.60 (1.4-1.7) 1.50 (1.3-1.7)

1.50 (1.4-1.6) 1.60 (1.4-1.9)

1.17 1.1-1.3) 1.40 1.3-1.5) 1.53 1.3-1.9)

0.50 (0.3-0.8) 1.30 (1.2-1.4) 1.60 (1.5-1.7)

0.80 (0.5-1.0) r 0.90 (0.8-1.0) 1.40 (1.4-1.4)

1.43 1.55 1.20 1.13 0.97 1.17

1.40 1.00 0.83 0.77 0.90 0.93

1.47 0.65 0.47 0.63 0.53 0.73

Isoflat'one Biochanin A

Flacan-3-ol Catechin Epicatechin

Flacanone Flavanone Naringenin

Flacone Apigenin Chrysin Flavone

Flaconol Flavonol Galangin Kaempferol Morin Myricetin Quercetin

(1.4-1.5) (1.3-1.8) (1.1-1.3) (1.1-1.2) (0.8-1.1) (1.1-1.3)

(1.3-1.5) (0.7-1.2) (0.7-1.0) (0.7-0.9) (0.8-1.0) (0.7-1.1)

(1.4-1.5) (0.6-0.7) (0.4 0.6) (0.5-0.7) (0.5-0.6) (0.6-0.8)

~ f r f J

a M N P C E (micronuclei in polychromatic erythrocytes) value of the control mice treated with corn oil only was 0.17 (0.1-0.2) per 100 PCE and the value from the positive control group treated with corn oil and B(a)P (150 m g / k g ) was 1.63 (1.4-2.0) per 100 PCE. All the flavonoid treated groups received B(a)P (150 m g / k g ) simultaneously and mice were sacrificed after 36 h as described in the text. . 1000 polychromatic erythrocytes were scored per animal. ~ Concentrations of flavonoid (mg/kg). d All data represent mean and range of at least 2 mice per group. e Significant dose-dependent reduction of MN compared to the positive control group ( p < 0.01; Analysis of Variance). p < 0.001.

247 TABLE 2 S U P P R E S S I O N O F B ( a ) P - I N D U C E D MNPCEs BY M U L T I P L E T R E A T M E N T O F F L A V O N O I D S IN B O N E - M A R R O W CELLS OF MICE a Flavonoids

Class

Chrysin Apigenin Galangin Kaempferol Morin Quercetin Myricetin Epicatechin

Flavone Flavone Flavonol Flavonol Flavonol Flavonol Flavonol Flavan-3-ol

M N P C E (and range) h 0.1 c

1.0

1.07 (1.0-1.1) 1.15 (1.1-1.2) 0.53 (0.4-0.6) 0.83 (0.8-1.0) 0.43 (0.4-0.5) 1.25 (1.2-1.3) 0.63 (0.6-0.7) 0.95 (0.9-1.0)

0.90 0.77 0.40 0.73 0.63 0.70 0.43 0.75

10.0 (0.8-1.(I) (0.7-0.8) (0.3-0.5) (0.6-0.9) (0.4-0.9) (0.6-0.8) (0.4-0.5) (0.7-0.8)

0.70 0.77 0.50 0.63 0.30 0.50 0.47 1.15

(0.6-0.8) (0.7-0.9) (0.4-0.6) (0.5-0.7) (0.3-0.3) (0.4-0.6) (0.4-0.5) (1.0-1.3)

" ~ ~ ~ ~ c c d

" M N P C E value of the control mice treated with corn oil (5 times) only was 0.17 (0.1-0.2) per 100 PCE and the value of the positive control group treated with corn oil (5 times) and B(a)P (150 m g / k g ) was 1.64 (1.5-2.0). All the flavonoid treated groups received flavonoids daily for 5 days and B(a)P (150 m g / k g ) on the last day. Mice were sacrificed after 36 h as described in the text. All data indicate m e a n (range) of at least two mice per group. b 1 000 polychromatic erythrocytes were scored per each animal. c Doses of flavonoid treated ( m g / k g / d a y , 5 days). d Significant dose-dependent reduction of MN compared to the positive control group ( p < 0.01; Analysis of Variance). p < 0.001.

suppression), even at the lowest dose level of 0.1 m g / k g / d a y (Table 2). As a control experiment, flavonoid derivatives were administered (10 mg/kg) without B(a)P injection to elucidate clastogenic activities of these compounds. Quercetin is the only one that induced a significant increase of MNPCEs (0.43 _+ 0.05) compared with the control group (0.17 _+ 0.05; p < 0.01, Table 3) under our experimental conditions. To investigate the anticlastogenic effect of flavonoids against different carcinogens, two non-clastogenic flavonoids were selected for further study. A potent anticlastogen, galangin, and a weak one, (-)-epicatechin, were used. As shown in Table 4, galangin showed an anticlastogenic effect against EMS- and DMBA-induced MNPCEs. On the other hand, (-)-epicatechin showed a weak anticlastogenic effect. However, both flavonoids did not show any anticlastogenic effect against ADM-induced MNPCEs. Discussion

It is generally accepted that flavonoids are not potent genotoxic agents (Carver et al., 1983; Van der Hoeven et al., 1984; Rueff et al., 1986; Popp and Schimmer, 1991), nor potent carcinogens

(Pamukcu et al., 1980; Saito et al., 1980; Morino et al., 1982) in eukaryotic systems. Several investigators have reported that some flavonoids are antimutagens (Huang et al., 1983; Francis et al., 1989; Wall et al., 1988), antipromoters (Nishino et ai., 1983; Yoshikawa et al., 1989) and anticarcinogen (Nixon et al., 1984). Since flavonoids are extensively consumed natural products, we have investigated the activities of 14 different kinds of flavonoids in mice. Our study shows that the frequencies of MNPCE which were induced by B(a)P in bone-marrow cells of mice were suppressed by the various flavonoid derivatives. All flavonoid derivatives showing anticlastogenic effects are found to have 5,7-dihydroxyflavone moiety with varying degrees of hydroxylations at the C-3 and B-rings. The flavone derivatives having 3,5,7-trihydroxyl groups appear to show higher anticlastogenic activity than the flavone derivatives having 5,7-dihydroxyl groups. This kind of structure-activity relationship is consistently observed after single or multiple treatment with the flavonoids. We have also shown that the 14 flavonoid derivatives are not clastogenic after treatment with 10 m g / k g of flavonoids. The only exception is quercetin which was not shown to be clasto-

248

genic by MacGregor et al. (1983) in mice. Our different exposure doses and conditions may account for the different observations. On the other hand, Popp and Schimmer (1991) reported that quercetin is genotoxic in human lymphocytes in vitro. When administered together with B(a)P, quercetin behaves like other nonclastogenic flavonoids. Therefore, the anticlastogenic effects of flavonoids appear to be independent of their own clastogenic activity. The anticlastogenic effects of flavonoids on other mutagens (EMS, DMBA, ADM) were also tested. We observed that the clastogenic effects of the first two but not ADM are reduced by galangin and (-)-epicatechin. From our study using a small group of chemicals, the data suggest that flavonoids may reduce the activities of only certain mutagens. Our observation is supported by other studies which indicate that some

TABLE 4 S U P P R E S S I O N OF EMS-, D M B A - A N D A D M - I N D U C E D M N P C E s BY G A L A N G I N A N D ( - ) - E P I C A T E C H I N a Group

Flavonoids

M N P C E (% and range) h

I s o f l a l 'oHf

Biochanin A

0.23 (0.2-0.3) c

Flaran-3-ol

Catechin Epicatechin

0.17 (0.1-0.2) 0.27 (0.2-0.3)

Flacanone

Flavanone Naringenin

0.13 (11.1-0.2) 0.17 (0.1-0.2)

Flat 'one

Apigenin Chrysin Flavone

0.30 (0.2-0.4) 0.13 (0.1-0.2) 0.20 (0.1-0.3)

Flat'onol

Flavonol Galangin Kaempferol Morin Myricetin Quercetin

0.20 0.13 0.17 0.20 0.33 0.43

(0.2-0.2) (0.1-0.2) (0.1-0.3) (0.1-0.4) (0.3-0.4) (0.4-0.5) d

~' M N P C E value of the control mice was 0.17 (0.1-0.2) per 100 PCE. All flavonoids were singly administered (10 mg/kg). b and c same as in Table 1. d Significant induction of MN compared to the control group ( p < 0.01; Students' t-test).

MNPCEs ~

Flavonoid Carcinogen (% and range) Control EMS DMBA ADM

400 100 5

0.17 (0.1-0.2) 1.20 (1.1-1.3) 1.47 (1.4-1.5) 0.87 (0.8-1.0)

Galangin + EMS

0.1 1.0 10.0

400 400 400

0.87 (0.8-1.()) 0.67 (0.6-0.7) 0.53 (0.5-0.6) c

Epicatechin + E M S

0.1 1.0 10.0

400 400 400

1.03 (0.9-1.2) 0.90 (0.8-1.0) 0.90 (0.8-1.0)

Galangin + D M B A

0.1 1.0 10.0

100 100 100

1.13 (1.0-1.3) 0.80 (0.5-1.0) 0.63 (0.5-0.9) c

Epicatechin+DMBA

0.1 1.0 10.0

1/11/ 100 100

0.97 (0.7-1.3) 1.50 (1.3-1.8) 1.23 (1.2-1.3)

Galangin + A D M

0.1 1.0 10.0

5 5 5

0.73 (0.5-0.9) 0.60 10.4-1.0) 0.70 (0.5-1.0)

Epicatechin + A D M

0.1 1.0 10.0

5 5 5

0.97(0.9-1.1) 1.07 (1.0-1.1) 1.00 (0.9-1.1 )

TABLE 3 C L A S T O G E N I C E F F E C T O F F L A V O N O I D S IN BONEM A R R O W CELLS O F MICE ~'

Dose ( m g / k g )

Each flavonoid was administered daily for 5 days. b Same as in Table 1. Significant dose-dependent reduction of MN compared to the positive control ( p < 0.01; Analysis of Variance).

flavonoids inhibit the production of D N A adducts by mutagens (Chang et al., 1985; Shah and Bhattacharya, 1986). In addition, flavonoids are also shown to interfere with metabolic activation of hydrocarbons that can form adducts on D N A (Sousa and Marietta, 1985; Kim et al., 1991). These and other mechanisms may be responsible for the anticlastogenic effects of flavonoids. In conclusion, we show that several flavonoids are potent anticlastogens in an in vivo system. In general, flavonoids that have been shown to be potent antimutagens in bacterial cells (e.g. kaempferol, morin, myricetin) are also demonstrated by us to be anticlastogens in mice. Their anticlastogenic effects appear to be unrelated to their own clastogenic activities. These compounds

249 also

seem

to

be

particularly

effective

against

chemicals that can form DNA adducts. This preferential activity appears to be due to their ability to interfere with metabolic activation of hydrocarbons and to block formation of DNA adducts. T h e r e f o r e , o u r in v i v o s t u d y s u g g e s t t h a t s o m e flavonoids may be anticarcinogens.

Acknowledgements This study was supported istry of Education

by the Korean Min-

(1989).

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