Anticlastogenic effects of galangin against mitomycin C-induced micronuclei in reticulocytes of mice

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Environmental Mutagenesis

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Mutation Research 360 (1996) 37-41

Anticlastogenic effects of galangin against mitomycin C-induced micronuclei in reticulocytes of mice Moon Young Heo a,*, Lee Hyung Jae a, Sohn Su Jung b, William W. Au c a College of Pharmacy, Kangweon National University, Chuncheon 200-701, South Korea b National Institute of Safety Research, 5 Nokbun-dong, Eunpyung-Ku, Seoul 122-020, South Korea c Department of Preuentive Medicine and Community Health, The University of Texas Medical Branch, Gah,eston, TX 77555-1110, USA Received 16 June 1995; revised 16 October 1995; accepted 31 October 1995

Abstract

We investigated the suppressive effect of galangin on the induction of micronucleated reticulocytes (MNRETs) by mitomycin C (MMC) in mouse peripheral blood. When galangin was given to mice 24 h before the intraperitoneal injection of MMC (1 mg/kg), a more marked decrease in the frequency of MNRETs was observed than in mice with simultaneous and post-treatment of galangin. On the other hand, when galangin was given to mice for 7 consecutive days before MMC injection, galangin showed potent anticlastogenic effects, even at the lowest dose level of 0.1 mg/kg. Results from our in vivo studies indicate that galangin is capable of suppressing the clastogenic activity of the direct acting MMC. Together with our earlier observations, it appears that galangin is capable of protecting cells from the toxic effects of a variety of hazardous chemicals. Therefore, galangin may be an useful chemopreventive compound. Keywords: Galangin; Anticlastogenic effect; Micronucleus test; Mitomycin C; Flavonoid

1. Introduction

Flavonoids, which are commonly found in plants, have anti-mutagenic and anti-carcinogenic activities against a number of genotoxic agents (Wattenberg and Leong, 1970; Francis et al., 1988; Huang et al., 1983). For example, they have suppressive effects against the mutagenic activities of several carcinogens in Salmonella typhimurium (Ogawa et al., 1985; Francis et al., 1988). We showed that a diverse group of flavonoids is anticlastogenic against the induction of micronuclei in bone-marrow cells of

* Corresponding author.

mice exposed to benzo[a]pyrene (Heo et al., 1992). The flavonol derivatives are particularly effective. Among them, galangin is very effective in suppressing the MNNG-induced micronuclei in bone-marrow cells and the bleomycin-induced chromosome aberrations in spleen cells of mice (Lee et al., 1993; Heo et al., 1993; Heo et al., 1994). On the other hand, it has been reported that galangin had strong inhibition (97%) of mutagenicity of 2-aminoanthracene on Salmonella typhimurium TA98 (Wall et al., 1988). The biological activities of galangin are substantiated by observations that it has scavenging activities (Waters et al., 1990) and that it is one of the most potent antioxidative flavonoids (Cholbi et al., 1991). Thus, galangin may potentially be an effective

0165-1161/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSDI 0 1 6 5 - 1 1 6 1 ( 9 5 ) 0 0 0 6 4 - X

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M. E Heo et al. / Mutation Research 360 (1996) 37-41

chemopreventive agent for several classes of chemical carcinogens. In this study, we investigated the suppressive effect of galangin on the induction of micronucleated reticulocytes (MNRETs) by mitomycin C (MMC) in mouse peripheral blood. MMC is a direct alkylating agent and an antitumor chemotherapeutic agent (Gebhart et al., 1980). However, its use in patients is limited by its toxicity to normal cells. Moreover, it is also carcinogenic (Dempsey et al., 1985). Therefore, the development of a protecting agent that can reduce the toxicity of MMC to normal cells may be helpful in improving the use of this drug. With this as one of our aims, we conducted a study to investigate the in vivo anticlastogenic activity of galangin on MMC-induce MNRETs.

2. Material and methods 2.1. Animals Male C3H mice (6 weeks, 20-25 g) obtained from the Animal Center of the Seout National University (Seoul, Korea) were used throughout the experiments. They were provided with free access to standard rodent chow and water, and maintained in a chamber with laminar air flow. The atmosphere in the chamber was maintained with a relative humidity of 55 + 7% and a temperature of 22 + I°C.

0.5-1.5 and 0.1-100 m g / k g , respectively, which were chosen by previous experiments (Heo et al., 1992, 1993). Mice were treated with MMC alone. with MMC and galangin simultaneously, with galangin for 24 h before treatment with MMC, or with galangin 24 h after treatment with MMC. In another protocol, mice were treated orally with galangin daily for 7 consecutive days, and then given MMC immediately after the last dose of galangin. Mice administered corn oil were used as vehicle controls. Peripheral blood samples from the treated animal were collected from a tail vein 48 and 54 h after treatment with MMC. In another experiment, mice were treated with different doses and for different times with MMC to evaluate a dose- and time-dependent effect. The micronucleus assay was carried out using the procedure of Hayashi et al. (1990). Briefly, 10 #1 of 1 m g / m l acridine orange (AO) aquous solution was spread on a pre-heated glass slide and air-dried. A 5 pA aliquot of blood sample was placed on an AOcoated glass slide, and then covered with a cover slip. For microscopic analysis, a combination of blue excitation (488 nm) and yellow-to-orange barrier filter (515 nm long pass) was used. One thousand reticulocytes (RETs) per animal were examined to determine the frequency of MNRETs for each treatment condition. There were 5 mice for each treatment condition. 2.4. Statistical analysis

2.2. Chemicals Galangin (548-83-4) was purchased from Aldrich Chemical Company (Milwaukee, USA), mitomycin (66F0494) was obtained from Sigma Chemical Company (St. Louis, MO, USA). Galangin was dissolved in corn oil for in vivo treatment. MMC was dissolved in distilled water. In all experiments, the administered volume to mice was 0.1 m l / 2 5 g of body weight. 2.3. Animal treatment and micronucleus test Five mice per dose were randomly assigned to each treatment group. MMC was intraperitoneally administered and galangin was administered orally. The administrative doses of MMC and galangin were

Analysis of Varience and Student's t-test were used for the statistical analysis.

3. Results 3.1. Time-course and dose-response study A summary of time-course study of MNRETs induced by MMC ( l m g / k g of body weight) is shown in Fig. 1. The maximum frequency of MNRETs was observed 48 h after the treatment. When galangin (10 m g / k g ) was orally administered together with MMC (1 m g / k g , i.p.), the MMC-induced MNRETs were reduced at every sampling time. As a control experiment, galangin (10 m g / k g ) was administered with-

M. Y. Heo et al. / Mutation Research 360 (1996) 37-41

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were chosen to determine the suppressive effect of galangin.

• MMC tmg/kg, i.p. v •

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MMC lmg/kg, i.p. + Galangin lOmg/kg, p.o. Galangin lOmg/kg, p.o.

3.2. Effects of galangin on MNRETs-induced by MMC The suppressive effects of galangin under different treatment conditions are shown in Table 1. In mice treated simultaneously with both chemicals, there were anticlastogenic effects in both the 48-h and 54-h sampling times. The MNRETs' frequency is significantly less than that from the MMC control when 1, 10, 100 m g / k g galangin were used in combination with MMC. In addition, the suppressive effect is more pronounced in the 54-h than the 48-h treatment groups (ANOVA test: p < 0.005 at 48 h, p < 0.001 at 54 h). When galangin was given to mice 24 h before the intraperitoneal injection of MMC (1 mg/kg), the most marked decrease in the frequency of MNRETs was observed (ANOVA test: p < 0.001 at 48 h, p < 0.001 at 54 h). Post-treatment with galangin showed limited suppressive effects against MMC-induced MNRETs at 48 h (ANOVA test: p = 0.07) and 54 h (ANOVA test: p < 0.05). When galangin was given to mice for 7 consecutive days and then immediately with MMC injection, galangin showed potent anticlastogenic activities,

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Time after treatment(h) Fig. I. Time-dependent effect of galangin on mitomycin C (MMC)-induced micronuclei in reticulocytes (MNRETs). Mice were treated with MMC alone ( O ) , MMC and galangin simultaneously ( v ) and galangin alone ( v ) and blood was collected from tail vein at various times for analysis. Values represent five mice _+SE.

out MMC injection to elucidate clastogenic activity of this compound. There was no increase in frequency of MNRETs up to 66 h after treatment. MMC showed a good dose-response induction of MNRETs caused by treatment with 0.5-1.5 m g / k g at 24, 48 and 72 h, respectively (data not shown). Therefore, in the subsequent study, the dose of 1 m g / k g of MMC and sampling times of 48 and 54 h

Table 1 Effects of various treatment with galangin on the frequency of MNRETs by MMC Galangin (mg/kg)

M N R E T s / 1 0 0 0 RETs a

48 h

0 0.1 1 10 100

54 h

Simul. b

Pre- c

Post- d

Multiple e

Simul. b

Pre- c

27.2 + 1.8 23.0±0.8 21.0±2.1 * 18.4±1.7 * 1 7 . 3 ± 1 . 2 **

22.3 _+ 1.9 17.0_+1.6 16.6+0.5 * 10.2-+1.7 ** 10.8-+1.9 **

24.2 -1- 1.6 21.5_+2.4 18.0±0.4 * 17.5±4.5 17.0_+1.7

20.7 ___ 1.4 11.4±1.4 9.2+0.7 11.8±0.7 9.3±1.5

24.0 ± 1.5 23.0_+1.1 1 3 . 4 ± 1 . 0 ** 18.0±1.4 * 1 1 . 3 ± 1 . 6 **

20.8 + 1.1 9 . 4 + 1.6 12.4+0.9 7 . 8 + 1.8 10.8+1.8

** ** ** **

** ** ** **

Post- a

Multiple e

18.3 + 1.8 17.0+1.7 14.3-+1.3 12.3+1.8 11.7+0.7 *

15.5 + 2.1 9.6+0.8 8.6+0.7 7.3_+1.1 4.3+1.8

* * * *

a 1000 RETs were analyzed per each animal. Peripheral blood collected 48 h and 54 h. b Simultaneous treatment: mice were treated with MMC(1 m g / k g , i.p.) and galangin simultaneously. c Pre-treatment: mice were treated with galangin at 24 h before treatment with MMC(1 m g / k g , i.p.). d Post-treatment: mice were treated with galangin at 24 h after treatment with MMC(1 m g / k g , i.p.). e Multiple pre-treatment: mice were treated with galangin daily for 7 consecutive days and then with M M C (1 m g / k g , i.p.) after the last dose of galangin. * '* * Significantly different from the control group at p < 0.05 and p < 0.01, respectively (Student's t-test).

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M. E Heo et al. / Mutation Research 360 (1996) 37-41

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Fig. 2. The suppressive effects of galangin on MMC-induced MNRETs under various conditions of treatment. Black bar: simultaneous treatment; open bar: pre-treatment; hatched bar: post-treatment: cross-hatchedbar: multiple pre-treatment. '" Significantlydifferent from the control group at p < 0.05 and p < 0.01, respectively (Student's t-test).

even at the lowest dose level of 0.1 m g / k g ( A N O V A test: p < 0 . 0 0 1 at 48 h, p < 0 . 0 0 1 at 54 h). All combined treatment conditions have significantly less MNRETs than the MMC control (Fig. 2). Although 5 mice were used in all experimental conditions, toxicity to reticulocytes was observed in the highdose groups because a limited number of reticulocytes was available for analysis from these mice.

4. Discussion The anticlastogenic effects of galangin against MMC using simultaneous, pre-and post-administration protocols were investigated. When a single dose of galangin was given to mice 24 h before the intraperitoneal injection of a single dose of MMC, the suppression in the frequency of MNRETs was more pronounced than the other two treatment conditions (Fig. 2). In addition, when galangin was administered in daily doses for 7 consecutive days, there were potent anticlastogenic effects, even at the lowest dose level of 0.1 m g / k g / d a y galangin (% suppression: 43.0% at 48 h and 38.1% at 54 h). Galangin itself has no effect on M N R E T frequencies. Based on the lower MNRETs frequency at 54 h compared with 48 h in MMC-exposed mice (Fig. 1), we expected that galangin should have more anticlastogenic effects at the earlier than the later harvest times. However, the data indicate more pronounced effects in most cases at 54 h (Fig. 2). Since the

difference between the 48-h and 54-h harvest groups is not consistent across all galangin treatment doses, the effect is probably not due to a simple delay of micronuclei formation in reticulocytes, but also to perturbation of entry of reticulocytes from bone marrow to the circulation. Some investigators have reported that the clastogenicity of MMC was inhibited by several classes of chemical including y-L-glutamyl-taurine (Toth and Csaba, 1988), tannic acid (Sasaki et al., 1990) and glutathione (Rita et al., 1991). Results from our in vivo studies also indicate that galangin is capable of suppressing the clastogenic activity of MMC. This phenomenon is similar to results reported by Wall et al. (1988), who reported that galangin inhibited 97e~ of the mutagenicity of 2-aminoanthracene in Salmonella typhimurium TA98. Together with other studies (Cholbi et al., 1991: Heo et al., 1993; He() el al., 1994), the data indicate that galangin may be an useful chemopreventive agent against damage from exposure to a variety of genotoxicants (e.g.. 2aminoanthracene, Neomycin, carbon tetrachloride. N-methyl-N'-nitro-N-nitrosoguanidine). Therefore, galangin may be an useful chemopreventive agent in vivo. In addition, pre-treatment with multiple doses of galangin appears to be more effective than posttreatment. We have not yet determined the mechanism which enables galangin to suppress the frequency of MNRETs induced by MMC in the present study. It is known that MMC is an alkylating and cross-linking

M.Y. Heo et al. / Mutation Research 360 (1996) 37-41 agent with D N A . Therefore, there are possibilities that the anticlastogenic effects o b s e r v e d in in v i v o m i c r o n u c l e u s assay m i g h t be due to the s c a v e n g i n g effect to react with alkyl radical or b l o c k i n g to do cross-linking b e t w e e n M M C and D N A although other possible m e c h a n i s m such as an e n h a n c e m e n t of D N A repair system c o u l d not be excluded. M o r e intensive research is required to find out the action m e c h a n i s m o f galangin against M M C .

Acknowledgements This research was supported in part by Grant 911-0460-012-2 f r o m the K o r e a Science and Engineering F o u n d a t i o n and a grant f r o m the N e w D r u g D e v e l o p m e n t Program, Ministry o f Health and Social Affairs. The authors are grateful to Mrs. Sandra R o o d for her preparation o f the manuscript.

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