Antimutagenic effects of cinnamaldehyde on chemical mutagenesis in Escherichia coli

Mutation Research, 107 (1983) 219-227

219

Elsevier Biomedical Press

Antimutagenic effects of cinnamaldehyde on chemical mutagenesis in Escherichia coli T. Ohta, K. Watanabe, M. Moriya and Y. Shirasu Institute of Environmental Toxicology, Suzuki-cho 2-772, Kodaira, Tokyo 187 (Japan)

T. Kada Department of Induced Mutation, National Institute of Genetics, Yata - 1111, Mishima, Shizuoka 411 (Japan)

(Received 29 January 1982) (Revision received 25 May 1982) (Accepted 1 June 1982)

Summary Antimutagenic effects of cinnamaldehyde on mutagenesis by chemical agents were investigated in Escherichia coli WP2 u v r A - t r p E - . Cinnamaldehyde, when added to agar medium, greatly reduced the number of Trp + revertants induced by 4-nitroquinoline 1-oxide (4-NQO) without any decrease of cell viability. This anti° mutagenic effect could not be explained by inactivation of 4-NQO caused by direct interaction with cinnamaldehyde. Mutagenesis by furylfuramide (AF-2) was also suppressed significantly. Mutations induced by methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS) were slightly inhibited. However, cinnamaldehyde was not at all effective on the mutagenesis of N - m e t h y l - N ' - n i t r o - N - n i t r o s o g u a n i d i n e ( M N N G ) . Two derivatives of cinnamaldehyde, cinnamyl alcohol and t r a n s - c i n n a m i c acid, did not have as strong antimutagenic effects on 4-NQO mutagenesis as cinnamaldehyde had. Because cinnamaldehyde showed marked antimutagenic effects against mutations induced by UV-mimic mutagens but not those induced by M N N G or EMS, it seems that cinnamaldehyde might act by interfering with an inducible error-prone D N A repair pathway.

All correspondenceshould be sent to: Toshihiro Ohta, Institute of EnvironmentalToxicology,Suzuki-cho 2-772, Kodaira, Tokyo 187, Japan. Abbreviations: AF-2, furylfuramide; EMS, ethyl methanesulfonate; 4-HAQO, 4-hydroxyaminoquinoline l-oxide; MMS, methyl methanesulfonate; MNNG, N-methyl-N'-nitro-N-nitrosoguanidine; 4-NQO, 4-

nitroquinoline l-oxide. 0027-5107/83/0000-0000/$03.00 © Elsevier Biomedical Press

220 As a result of current progress and improvement in testing methods for detecting mutagens, it has become clear that a great number of chemical mutagens, including artificial and natural products, exist in our environment. In evaluating the mutagenicity of environmental mutagens, it may also be important to know about factors that affect mutagenic activity in our environment. A number of antimutagenic factors working in bacteria have long been known. (See Clarke and Shankel, 1975.) It is only recently that our attention has become focused on environmental antimutagens. The antimutagenic activity of cinnamaldehyde was originally observed on UV-induced mutagenesis in E. coli B / r WP2 strain as a result of systematic screening for antimutagenic factors in foodstuffs (Kada, Ikegawa and Nomoto, unpublished observation). In this paper, we report the antimutagenic effects of cinnamaldehyde on mutagenesis by 4-NQO and other chemical mutagens in E. coli. Cinnamaldehyde ( C 6 H s C H = C H C H O ) , a yellowish oily liquid with a strong odor of cinnamon, is a main component of Ceylon and Chinese cinnamon oils found in bark of Cinnamomum zeylanicum and Cinnamomum cassia. It has been used in the flavor and perfume industry.

Materials and methods Chemicals

Cinnamaldehyde (CAS 104-55-2), cinnamyl alcohol (CAS 104-54-1), trans-cinnamic acid (CAS 621-82-9), 4-hydroxyaminoquinoline 1-oxide (CAS 4637-56-3) and 4-nitroquinoline 1-oxide (4-NQO, CAS 56-57-5) were purchased from Tokyo Kasei Kogyo Co., Tokyo. Methyl methanesulfonate (MMS, CAS 66-27-3) and ethyl methanesulfonate (EMS, CAS 62-50-0) were obtained from Eastman Kodak Co., New York. N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG, CAS 70-25-7) was obtained from Wako Pure Chemical Industries, Osaka. Bacterial strain and media WP2 uvrA- t r p E - , obtained from Dr. E.M. Witkin, was used throughout this

study. The bacteria were cultured in Nutrient Broth No. 2 (Oxoid). For reverse mutation assays, semi-enriched minimal (SEM) agar medium was used (Witkin, 1975). The SEM agar medium consisted of Vogel-Bonner E medium (2 g citric acid monohydrate, 10 g K 2 H P O 4, 3.5 g N a ( N H a ) H P O 4 • 4H20, 0.2 g MgSO 4 • 7HzO per 1) supplemented with 0.4% glucose, 1.5% agar (Difco) and 5% (v/v) liquid nutrient broth (8 g BBL nutrient broth and 5 g NaC1 per 1). Cinnamaldehyde and its derivatives were dissolved in a small amount of dimethylsulfoxide (DMSO), diluted with distilled water and then added to the autoclaved SEM agar medium just before the plating. The final concentration of DMSO in a SEM agar plate was less than 0.1%. All plates were used for experiments within 4 days after preparation. Reverse mutation assays

0.5 ml of a bacterial overnight culture was inoculated into 5 ml of fresh nutrient

221 broth and incubated at 37°C with shaking for 180 min. The bacteria in log phase (about 2 x 109/ml) were washed twice and resuspended in 1/15 M phosphate buffer (pH 7.2). A mutagen dissolved in distilled water or DMSO was added into the bacterial suspension, and bacteria were treated for 60 min at 37°C, unless otherwise noted. After the treatment, cells were washed twice with buffer by centrifugation and resuspended in buffer. For the detection of Trp ÷ revertants, 0.1-ml portions of the bacterial suspension were spread on SEM plates containing cinnamaldehyde at various concentrations or on control plates with no cinnamaldehyde. The bacterial suspension was diluted 106-fold, and 0.1-ml aliquots were spread on the same SEM plates to allow determination of the cellular viability. After 2 days' incubation at 37°C, revertants and viable cells were counted. All assays were performed 2 - 4 times and all values reported are the averages of triplicate plates. Isolation of Trp + -revertant colonies and susceptibility to cinnamaldehyde

4-NQO-induced revertant colonies on a SEM plate without cinnamaldehyde were picked up randomly and purified on tryptophan-free minimal agar plates by single-colony isolation. These purified Trp + clones were then cultured in liquid minimal medium for 1 day. After appropriate dilutions, about 100 cells of each culture were spread on SEM plates containing cinnamaldehyde at various concentrations, and the plates were incubated for 1-2 days at 37°C to test their colony-forming abilities in the presence of cinnamaldehyde. Characterization of Trp + revertants

An ochre mutant of phage T4 was used to investigate the contribution of sup + mutations to the total number of phenotypically Trp ÷ revertants. T4 ochrel9 mutant phage was a gift from Dr. B.A. Bridges (Brighton). This T4 mutant phage can multiply only in host bacteria containing a suppressor mutation for the ochre codon. 100 Trp ÷ revertant colonies, each induced by 4-NQO or MNNG, were isolated and purified. Phage lysate (5 x 107/ml) was streaked on peptone agar plates (1% peptone, 0.5% NaCI, 1% agar, pH 7.2). Then an overnight culture of each bacterial clone was cross-streaked and the plates were incubated for 20 h at 37°C. Bacterial lysis at the cross region was taken as evidence of phage growth, indicating suppression.

Results

Effects of cinnamaldehyde on 4-NQO mutagenesis and cellular viability are shown in Tables 1 and 2. Cinnamaldehyde at concentrations increasing from 10 to 50 ttg/ml in SEM agar plates reduced the number of Trp ÷ revertant colonies, whereas no decrease was found in cellular viability and the number of spontaneous revertants. About 85% reduction in the number of revertants induced by 4-NQO was observed on the SEM plate containing cinnamaldehyde at 40 # g / m l (Table 1). Furthermore, the presence of cinnamaldehyde at a concentration of 40 t t g / m l in the medium resulted in a significant reduction in the number of revertants at all the

222 TABLE 1 A N T I M U T A G E N I C EFFECTS OF C 1 N N A M A L D E H Y D E ON 4-NQO M U T A G E N E S I S IN E. coli WP2 u v r A - t r p E 4-NQO: 2 # g / m l

4-NQO: 0 p.g/ml

Cinnamaldehyde in medium ( # g / m l )

Revertants per plate

Viable cells per plate

Revertants per plate

Viable cells per plate

0 10 20

6 7 5

193 191 184

816 684 358

127 140 140

30 40 50

6 6 7

201 183 188

206 122 113

142 153 144

Cells were treated with 4-NQO for 60 rain at 37°C.

concentrations of 4-NQO that were tested (Table 2). Though 4-NQO was removed by washing before plating on cinnamaldehyde-containing SEM plates, the possibility that cinnamaldehyde might inactivate 4-NQO incorporated into bacterial cells by direct interaction could not be completely neglected. To study this possibility, bacterial cells were treated with 4-NQO in the presence of cinnamaldehyde for 60 min. Treated cells were washed twice and plated onto cinnamaldehyde-free SEM plates. As shown in Table 3, a high level of mutation induction was still observed in as many as in the cinnamaldehyde untreated control. This result indicates that cinnamaldehyde does not inactivate the mutagen directly like a 'desmutagen'. 4-NQO is metabolically activated to 4-hydroxyaminoquinoline 1-oxide (4-HAQO) by bacterial nitroreductase, and then 4-HAQO is bound to purine residues of DNA through catalysis by seryl-tRNA synthetase (Tada and Tada, 1976). To investigate

TABLE 2 A N T I M U T A G E N I C EFFECTS OF C I N N A M A L D E H Y D E ON 4-NQO MUTAGENES1S 4-NQO (p,g/ml)

0 0.5 1

2 3

Cinnamaldehyde: 0 # g / m l medium

Cinnamaldehyde: 40 ~tg/ml medium

Revertants per plate

Revertants per plate

Viable cells per plate

Viable cells per plate

10 28

184 162

8 11

183 169

87 685 1060

190 109 37

27 137 292

194 130 66

Cells were treated with 4 - N Q O f o r 6 0 m i n at 37°C.

223 TABLE 3 EFFECTS OF C I N N A M A L D E H Y D E IN T H E T R E A T M E N T M E D I A ON S P O N T A N E O U S A N D 4-NQO-INDUCED MUTATION 4-NQO (/~g/ml)

Cinnamaldehyde (~g/ml)

Revertants per plate

Viable cells per ml

0 0

0 40

5 4

2.0 x 109 1.7 x 109

2 2 2

0 20 40

737 755 712

1.5 x 109 1.4× 109 1.4x l09

Cells were treated with 4-NQO in the presence or absence of cinnamaldehyde in phosphate buffer for 60 min, washed twice, and then plated on SEM plates.

the possible inhibitory effects of cinnamaldehyde on the metabolic activation of 4-NQO in vivo, 4-HAQO was tested. In 4-HAQO mutagenesis, reduction of Trp ÷ revertants equal to that in 4-NQO mutagenesis was observed (data not shown). Therefore, the decrease in the number of revertants induced by 4-NQO could not be explained by an inhibitory action of cinnamaldehyde at least on the nitroreductase. The possibility that the growth of fixed mutants might be preferentially inhibited was also suspected, because cinnamaldehyde was present throughout the period of colony formation of Trp ÷ cells. Therefore, 50 clones of 4-NQO-induced revertants were isolated randomly from the cinnamaldehyde-free SEM plate and tested for colony-forming ability on the cinnamaldehyde-containing SEM plates. All the clones had colony-forming ability and grew well on the SEM plates containing cinnamaldehyde (20, 40 and 60 ffg/ml). Thus, cinnamaldehyde does not inhibit the cell growth of Trp ÷ revertants. Further, we investigated whether derivatives of cinnamaldehyde had antimutagenic effects on 4-NQO mutagenesis. As shown in Fig. 1, cinnamic acid (C6HsCH=CHCOOH) was scarcely effective on 4-NQO mutagenesis, while cinnamyl alcohol (C6HsCH~--~CHCH2OH) was weakly effective compared with cinnamaldehyde. The antimutagenic effect of cinnamyl alcohol might be due to some cinnamaldehyde possibly converted metabolically from cinnamyl alcohol in the bacterial cells. Finally, the effects of cinnamaldehyde on mutations induced by other mutagens were tested (Fig. 2). Bacterial cells pretreated with AF-2, MNNG, MMS or EMS were spread onto the SEM plates containing various concentrations of cinnamaldehyde in the same way as for 4-NQO mutagenesis. Mutations induced by AF-2 were depressed about 75% on the SEM plate containing cinnamaldehyde at 40/~g/ml. MMS- and EMS-induced mutations were slightly inhibited. About 28 and 23% reductions were observed, respectively, on the plates with 40/tg of cinnamaldehyde per ml. In contrast, cinnamaldehyde was not effective against M N N G mutagenesis at all.

224 300

'~

200

100

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0 60O

600

60C @

o. 40C

400

40(

2OO

200

20C

>

ID

0

5

100

1'0

150

Clnnamyl alcohol ( )Jg/ml )

2'0

310 410

0

Clnnamaldehyde ( pg/ml )

5

~

'

100

,

150

Clnnamlc acid ( ~g/ml )

Fig. 1. C o m p a r i s o n of a n t i m u t a g e n i c effects of c i n n a m a l d e h y d e and its derivatives on 4 - N Q O mutagenesis. Cells were treated with 4 - N Q O (2 p , g / m l ) for 60 min at 37°C.

700

300 200

600 q

100

50O 400

700

30O

600

2O0 •

IO0

~. ~. te

0

800

500 AF-2 1'0

MNNG

2'0

30

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0

Q

1'0

2'0

80

4JO

~" "x :300 ~

• ~=

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400

300'

300

200

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100

100

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110 2()

30

40

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EMS

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210

310 410

Clnnamaldehyde ( )Jg/ml ) Fig. 2. Effects of c i n n a m a l d e h y d e on m u t a t i o n i n d u c t i o n s in E. coli W P 2 uvrA - t r p E - p r e t r e a t e d with A F - 2 (0.4 # g / m l ) , M N N G (2 # g / m l ) , M M S (1.2 m g / m l ) or EMS (1 m g / m l ) for 60 min.

225 TABLE4 CHARACTERIZATION OF Trp + REVERTANTCLONES IN T4 OCHRE MUTANT PHAGE Mutagen

4-NQO (2 #g/ml) MNNG (2 #g/ml)

Number of clones examined/ total revertants induced

Phage growth

Suppressor mutation (%)

[+ ] ( mutationSUppress°r'

[-]

100/627

94

6

94

100/825

92

8

92

trpE gene mutation )

The WP2 strain has an ochre mutation in one of the genes for tryptophan biosynthesis. Tryptophan-independent revertants (Trp +) appear either by a base change at the site of the original alteration (trp ÷ mutation) or by a base change at other sites in the chromosome (t-RNA genes), a change that suppresses the original alteration (sup ÷ mutation) (Bridges et al., 1967; Osborn and Person, 1967). The absence of antimutagenic effects on M N N G mutagenesis could not be explained by the differential types of mutation in 4-NQO mutagenesis and M N N G mutagenesis, as shown in Table 4. In both cases (4-NQO and M N N G mutagenesis), the majority of Trp ÷ revertant clones had sup ÷ mutations (94 and 92%, respectively).

Discussion

The factors usually called 'antimutagens' may be classified into three categories. 1. Desmutagens are factors that inactivate or destroy mutagens. (See Kada et al., 1982, for review.) For example, tryptophan pyrolysate mutagens (Sugimura et al., 1977) are enzymatically oxidized by peroxidase in cabbage or in human myeloma cells, and lose their mutagenic activities (Kada et al., 1978; Morita et al., 1978; Yamada et al., 1979; Inoue et al., 1981). The pesticide captan may react with sulfhydryl group(s) in the $9 fraction, blood and cysteine, and in consequence the mutagenicity disappears (Moriya et al., 1978). 2, Metabolism inhibitors are inhibitory factors in metabolic activation of promutagens. For example, vitamin A and selenium inhibit the activation, respectively, of 2-fluorenamine and 7,12-dimethylbenz[a]anthracene to metabolic forms that are mutagenic in Salmonella typhimurium TA98 (Baird and Birnbaum, 1979; Martin et al., 1981). 3. 'Antimutagens' in a narrow sense. A number of factors are known that interfere with spontaneous or induced mutagenesis at a cellular level. In this communication, we observed that cinnamaldehyde acted as an antimutagen at the cellular level by interfering with the cellular mutagenesis induced by 4-NQO or AF-2 but not by EMS or M N N G . A t least two types of mutagen are

226

known as to dependence on, or independence on the recA gene function for induction of mutations: 4-NQO and AF-2 initiate mutations belonging to the former type, and MNNG and EMS the latter (Witkin, 1969; Kondo et al., 1970). Researches for the role of the recA gene have indicated that an inducible error-prone repair that takes part in mutation induction (SOS function: Radman, 1975; Witkin, 1976) might involve DNA polymerase III (Bridges and Mottershead, 1978). Further, the functional umuC gene, which is involved in the SOS pathway of mutagenesis (Kato and Shinoura, 1977), is not required for mutation induction by EMS or MNNG (Schendel and Defais, 1980). EMS induces mutations in prophage X without affecting prophage induction (Moreau and Devoret, 1977). This lysogenic induction is one expression of the SOS functions. Therefore, EMS mutagenesis in ~,-lysogens is not a consequence of the induction of the SOS functions. Mutation induction by AF-2 was markedly enhanced by introduction of the hcr character into E. coli B/r WP2 in a manner similar to that with UV (Kondo and Ichikawa-Ryo, 1973; Kada, 1973). Cinnamaldehyde showed marked antimutagenic effects against mutations induced by UV-mimic mutagens such as 4-NQO and AF-2 but not those induced by MNNG or EMS. Therefore, one possible explanation is that cinnamaldehyde interferes with an inducible error-prone DNA repair system.

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227 prophage )~ in Escherichia coli KI2, in: H.H. Hiatt, J.D. Watson and J.A. Winsten (Eds.), Origins of Human Cancer, Cold Spring Harbor Laboratory, University of Tokyo Press, pp. 1451-1472. Morita, K., M. Hara and T. Kada (1978) Studies of natural desmutagens: Screening for vegetable and fruit factors active in inactivation of mutagenic pyrolysis products from amino acids, Agric. Biol. Chem., 42, 1235-1238. Moriya, M., K. Kato and Y. Shirasu (1978) Effects of cysteine and a liver metabolic activation system on the activities of mutagenic pesticides, Mutation Res., 57, 259-263. Osborn, M., and S. Person (1967) Characterization of revertants of E. coli WU36-10 and WP2 using amber mutants and an ochre mutant of bacteriophage T4, Mutation Res., 4, 504-507. Radman, M. (1975) SOS repair hypothesis: Phenomenology of an inducible DNA repair which is accompanied by mutagenesis, in: P.C. Hanawalt and R.B. Setlow (Eds.), Molecular Mechanisms for Repair of DNA, Plenum, New York, pp. 355-367. Schendel, P.F., and M. Defais (1980) The role of umuC product in mutagenesis by simple alkylating agents, Mol. Gen. Genet., 177, 661-665. Sugimura, T., T. Kawachi, M. Nagao, T. Yahagi, Y. Seino, T. Okamoto, K. Shudo, T. Kosuge, K. Tsuji, K. Wakabayashi, Y. litaka and A. ltai (1977) Mutagenic principle(s) in tryptophan and phenylalanine pyrolysis products, Proc. Jpn. Acad., 53, 58-61. Tada, M., and M. Tada (1976) Metabolic activation of 4-nitroquinoline I-oxide and its binding to nucleic acid, in: P.N. Magee, S. Takayama, T. Sugimura and T. Matsushima (Eds.), Fundamentals in Cancer Prevention, University of Tokyo Press, pp. 217-228. Witkin, E.M. (1969) Ultraviolet-induced mutation and DNA repair, Annu. Rev. Microbiol., 23, 478-514. Witkin, E.M. (1975) Persistence and decay of thermoinducible error-prone repair activity in nonfilamentous derivatives of tif-1 Escherichia coli B/r: The timing of some critical events in ultraviolet mutagenesis, Mol. Gen. Genet., 142, 87-103. Witkin, E.M. (1976) Ultraviolet mutagenesis and inducible DNA repair in Escherichia coli, Bacteriol. Rev., 40, 869-907. Yamada, M., M. Tsuda, M. Nagao, M. Mori and T. Sugimura (1979) Degradation of mutagens from pyrolysates of tryptophan, glutamic acid and globulin by myeloperoxidase, Biochem. Biophys. Res. Commun., 90, 769-776.