Mutation Research, 243 (1990) 299-302

299

Elsevier MUTLET 0329

Suppressing effects of vanillin, cinnamaldehyde, and anisaldehyde on chromosome aberrations induced by X-rays in mice Yfi F. S a s a k i , T o s h i h i r o O h t a , H i s a k o I m a n i s h i , M i e W a t a n a b e , Tomoko Kato and Yasuhiko Shirasu

Kyomu

Matsumoto,

Institute of Environmental Toxicology, Suzuki-cho 2-772, Kodaira, Tokyo 187 (Japan)

(Accepted 23 November 1989)

Keywords: Vanillin; Cinnamaldehyde;p-Anisaldehyde;Suppressingeffecton micronuclei;X-rays

Summary X-ray-induced chromosome aberrations were suppressed when vanillin, cinnamaldehyde, or p-anisaldehyde was given orally to mice after X-ray irradiation. Chromosome aberrations were monitored by the occurrence of polychromatic erythrocytes with micronuclei in bone marrow cells. The frequency of micronuclei was depressed about 55-6007o without toxicity of the test compounds to the bone marrow.

In the past several years, we have studied the antimutagenic and anticlastogenic effects of naturally occurring compounds in bacteria (Ohta et al., 1983a,b,c, 1986, 1988) and cultured mammalian cells (Sasaki et al., 1987a,b, 1988, 1989a,b, 1990; Imanishi et al., 1990). These compounds include vanillin, cinnamaldehyde and p-anisaldehyde which are used widely as flavorings in confectionery, beverages and foodstuffs. To know the antimutagenic activity of these flavorings in the whole mammalian system is more important to evaluate the adverse effects of environmental mutagens to our health. Recently, we reported on the suppressing effects of vanillin on chromosome aberrations and mutations induced by chemical mutagens such as mitomycin C and ethylnitroCorrespondence: Yfi F. Sasaki, Institute of Environmental Toxicology,Suzuki-cho2-772, Kodaira, Tokyo 187 (Japan).

sourea in mice (Inouye et al., 1988; Imanishi et al., 1990). In the present study, our attention is focused on chromosome aberrations induced by X-rays. We demonstrate the suppressing effect of vanillin, cinnamaldehyde and p-anisaldehyde using the mouse micronucleus test which is a well established and sensitive method to detect chromosome aberrations in bone marrow cells.

Materials and methods Vanillin, cinnamaldehyde, and p-anisaldehyde were purchased from Tokyo Kasei Kogyo (Tokyo, Japan). Vanillin was suspended in a 2°7o solution of Tween 80 (Wako Pure Chemical Industry, Osaka, Japan), and the others were dissolved in olive oil (Kosakai Seiyaku Co., Tokyo, Japan). The administration volume of the compounds was 10 ml/kg. Male ddY mice at 8 weeks of age were pur-

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chased from J a p a n SLC lnc. ( H a m a m a t s u , Japan). Four mice were assigned to each experimental group. Mice were irradiated with X-rays at 200 rad using a Hitachi X-ray machine with an adjusted Victreen dosimeter. After irradiation each flavoring was administered orally at 250, 313 or 500 m g / k g . In all experiments except for the time-course study, bone m a r r o w cells were sampled 24 h after the irradiation. In the time-course study, each flavoring at 500 m g / k g was given to mice immediately after the irradiation and bone marrow cells were sampled periodically. The micronucleus assay was performed according to the methods described by Schmid (1976). The frequencies of polychromatic erythrocytes with micronuclei (MNPCEs) per 1000 polychromatic erythrocytes (PCEs), and PCEs per 1000 red blood cells per mouse were determined. For statistical analysis, Student's t-test was used to compare the treated and untreated groups.

FABLE EFFECT

I OF

DIFFERENT

POST-TREATMENT INTERVALS

MICRONUCLE1 Interval

INDUCED

ON

WITH THE

VANILLIN

AT

FREQUENCY

OF

BY X - R A Y S

Frequency of MNPCEs

Frequency of

(h)

PCEs (%) M e a n _+ SD (%0)

Relative v a l u e ~

M e a n _+ SD

0

1.55 + 0.47**

0.45

54.4 +

3

1.83 _+ 0.26**

0.53

54.6 _+ 4.01

6

1.90 _+ 0.24**

0.55

59.8 _+

9

1.95 + 0 . 7 1 "

0.57

52.6 +_ 4.03

12

2.30 + 0.71

0.67

48.8 +

15

3.52 _+ 0.55

1.02

58.3 + 10.5

18

3.31 _+ 0.31

0.96

51.1 +

3.03 6.84 5.34 7.01

Control a

3.45 _+ 0.71

1.00

56.7 _+ 4.75

b

0.03 _+ 0.06

0.01

54.2 _+

7.12

B o n e m a r r o w cells w e r e p r e p a r e d 24 h a f t e r the i r r a d i a t i o n . a X - r a y (200 r a d ) c o n t r o l g r o u p w i t h o u t vanillin t r e a t m e n t . b Vanillin (500 m g / k g ) c o n t r o l g r o u p w i t h o u t i r r a d i a t i o n . ' R e l a t i v e value is c a l c u l a t e d f r o m the m e a n f r e q u e n c y d i v i d e d b y t h a t o f the X - r a y c o n t r o l g r o u p .

Results and discussion

S i g n i f i c a n t d e c r e a s e : * 0.01 < p < 0 . 0 5 ;

** 0.001 < p < 0 . 0 1 .

To find out the most sensitive period for flavoring treatment, vanillin was given to mice at 500 m g / k g every 3 h after X-ray irradiation at 200 tad. The most pronounced reduction in the frequency of M N P C E s was observed when vanillin was administered to mice just after the irradiation. The frequency of M N P C E s was decreased by 55% (Table 1). Significant suppression by vanillin was oberved until 9 h after irradiation. In the following experiments, therefore, the test compounds were given to mice immediately after the irradiation. Table 2 shows the dose-dependent decreases in M N P C E s by vanillin, cinnamaldehyde, and panisaldehyde. At the highest dose of 500 m g / k g , 55, 58 and 60% decreases were observed, respectively. To investigate whether these compounds cause a delay in the formation of micronuclei by their toxic effects, a time-course study was conducted. As shown in Fig. 1, the frequency of M N P C E s was reduced by the administration of each flavoring at any sampling time, and no delay in the induction of M N P C E s was observed. Furthermore, these compounds did not affect the fre-

quency of PCEs (Tables 1 and 2). These results indicate that the observed reduction of M N P C E s was not a reflection of the toxic effect of the flavorings to the bone marrow. In our previous paper, we showed that micronucleus induction by mitomycin C was suppressed by post-treatment with vanillin 6 - 9 h after the intraperitoneal injection of mitomycin C (Inouye et al., 1988). On the other hand, X-ray-induced micronuclei were suppressed when vanillin was given to mice 0 - 9 h after irradiation. Considering the kinetics of micronucleus formation and erythropoesis described by Salamone and Heddle (1983), X-ray-irradiated mice were sensitive to the action of vanillin throughout the last blast stage, while vanillin seemed to affect bone marrow cells treated with mitomycin C during a relatively short period just before the last mitosis. X-ray irradiation causes strand breaks in DNA. D N A adducts formed by mitomycin C are thought to convert into DNA-strand breaks in the S phase of the cell cycle (see Bender, 1979, for review). These DNA-strand

301

TABLE 2 SUPPRESSING EFFECTS OF VANILLIN, CINNAMALDEHYDE, AND p-ANISALDEHYDE ON THE FREQUENCY OF MICRONUCLEI INDUCED BY X-RAYS Flavoring

Mean ± SD (070)

Relative valuea

Frequency of PCEs (070) Mean _+ SD

3.35 ± 0.48

1.00

56.4 ± 4.16

Frequency of MNPCEs

(mg/kg)

Control

0

Vanillin

250 313 500

1.93 ± 0.26** 1.95 + 0.42** 1.50 + 0.39***

0.58 0.58 0.45

51.5 ± 3.38 51.7 +_ 4.61 51.1 ± 5.80

Cinnamaldehyde

250 313 500

3.38 _+ 0.38 2.67 _+ 0.67 1.40 + 0.47**

1.01 0.80 0.42

54.3 _+ 7.42 57.5 ± 7.19 52.7 ± 6.21

Anisaldehyde

250 313 500

2.00 + 0.20** 1.73 _+ 0.15"* 1.33 + 0.21"**

0.60 0,52 0.40

45.1 ± 6.73 51.3 ± 4.15 52.7 ± 4.38

Each flavoring was given orally to mice immediately after irradiation at 200 rad. Bone marrow cells were prepared 24 h after the irradiation. a Relative value is calculated from the mean frequency divided by that of the X-ray control group. Significant decrease: ** 0.001