The effect of quercetin on the mutagenicity of 2-acetylaminofluorene and benzo[a]pyrene in Salmonella typhimurium strains

Mutation Research, 142 (1985) 103-107

103

Elsevier

MRLett 0652

The effect of quercetin on the mutagenicity of 2-acetylaminofluorene and benzo[a]pyrene in Salmonella typhimurium strains Shunjiro Ogawa, Teruhisa Hirayama, Motoshi Nohara, Mitsuo Tokuda, Kunio Hirai and Shozo Fukui Kyoto Pharmaceutical University, Nakauchi-cho 5, Misasagi, Yamashina-ku, Kyoto 607 (Japan) (Accepted 5 November 1984)

Summary The comutagenic and desmutagenic effect of quercetin on the mutagenicity of typical mutagens e.g. 2-acetylaminofluorene (AAF), 4-nitroquinoline-l-oxide (4NQO) and benzo[alpyrene (B[a]P), in Salmonella typhimurium TA98, TA100 and TA98/1,8 DNP6 were examined. In the mixed application of AAF with quercetin in the presence of mammalian metabolic activation system ($9 mix), the numbers of revertants in TA98 increased by as much 2.2-5.0-fold compared with the sum of those in the separate applications of AAF and quercetin. A 1.4-2.7-fold increase was observed in TA100. Quercetin did not affect the mutagenicity of 4NQO, and depressed that of B[a]P. Dose-response curves for mutagenicity of quercetin with or without AAF (5/~g/plate) were examined. The results suggest that quercetin, present in a molarity of up to 1.5 times that of AAF, is apparently effective in enhancing the mutagenicity of AAF, because a linear dose-response curve was observed in the range of 0-5 ~g/plate quercetin with AAF although quercetin alone was not mutagenic in the same range. Dose-response curves for mutagenicity of quercetin with or without 5 ug/plate B[a]P did not increase compared with that for quercetin alone. The mutagenicity of the mixed application of B[a]P with quercetin was reduced to about 60°7o of the sum of separate application at doses ranging from 25 to 100/~g/plate of quercetin. Since enhancement and depression of mutagenicity by quercetin were observed for indirect mutagens, AAF and B[a]P, respectively, in the presence of $9 mix, quercetin may affect the metabolic pathway of these mutagens.

Quercetin (3,3' ,4' ,5,7-pentahydroxyflavon), one of the most popular flavonoids occurring in many plants (Robinson, 1967), is known to be mutagenic in Salmonella typhimurium TA98 and TAI00 with or without mammalian metabolic activation system ($9 mix) (Sugimura et al., 1977; Bjeldanes et al., 1977; Brown et al., 1979). However, the carcinogenicity of quercetin is still

doubtful although a few reports suggest that quercetin causes intestinal and bladder tumors in mice and rats (Boyland et al., 1964; Pamukcu et al., 1980). Because flavonoids, including quercetin, are consumed from foods of plant origin such as vegetables, fruits, crops and tea (Herrmann, 1976), it is of interest to determine whether flavonoids have a comutagenic or

0165-7992/85/$ 03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

104 desmutagenic effect on other mutagens or carcinogens. This report describes the effect of quercetin on the mutagenicity of the typical mutagens, 2-acetylaminofluorene (AAF), 4-nitroquinoline-1oxide (4NQO) and benzo[a]pyrene (B[a]P), which were used as amino, nitro and polycyclic hydrocarbon compounds, respectively, in Salmonella typhimurium tester strains.

3000

2000

i00o

Materials and m e t h o d s

Chemicals Quercetin, AAF, 4NQO, dimethyl sulfoxide (DMSO) and glucose 6-phosphate (G6P) were purchased from Nakarai Chemicals Co. Ltd., Kyoto (Japan), and B[a]P from Wako Pure Chemical Industries Co. Ltd., Osaka (Japan). All these reagents were guaranteed grades. N A D P H was purchased from Kohjin Co. Ltd., Tokyo (Japan) and glucose-6-phosphate dehydrogenase (G6PD) from Sigma Chemical Co. Ltd., St. Louis, MO (USA).

i

I

50

i00 $9

i 150

I 200

~ 250

i 300

(~i/plate)

Fig. 1. Effect of $9 content in $9 mix on mutagenicityof mixed application of AAF with quercetin in TA98. The test mixture contained 0.1 ml of DMSO test solution containing 5 ~g/plate AAF with 10 #g/plate quercetin, 0.5 ml of $9 mixture and 0.1 ml bacterial culture, and was preincubated for 20 min at 37°C. Revertants were counted after 48 h incubation at 37°C. Numbers of spontaneous revertants in DMSO with $9 mix (56+_5, 53+6, 57_+4, 49_+7, 55_+6, 84+_7, 33_+3, 29_+5 and 25_+7 in 5, 10, 20, 50, 100, 150, 200, 250 and 300/zl S9/plate, respectively) were subtracted. Each point is the mean value of 9 determinations.

Metabolic activation system ($9 mix) The postmitochondrial fraction ($9) was prepared from the livers of male Sprague-Dawley rats induced with PCB (pentachloro). A homogenate (3 ml of 0.15 M KC1/g liver) was centrifuged as described (Ames et al., 1975). Preparation of the $9 mix was performed according to Yahagi (1977) so that it contained 8 /zmoles MgCI2, 33 #moles KCI, 5/zmoles G6P, 4/zmoles N A D P H , 0.5 unit G6PD, 100 #moles sodium phosphate buffer (pH 7.4), water and 200 #1 $9 in a total volume of 1 ml.

Mutagenicity test Mutagenicity tests were performed essentially by the Ames method (Ames et al., 1975; Marson and Ames, 1983) with suspension assay as modified by Yahagi (1977). Samples were dissolved in DMSO. The assay was performed in the absence or presence of $9 mix to simulate mammalian metabolism. Every assay was carried out in triplicate on 3 separate occasions.

Results and discussion Fig.1 shows the mutagenicity in TA98 of mixed application of A A F (5 #g/plate) with quercetin (10 ~g/plate) assayed with a varying $9 content (#l/plate). The maximum number of revertants was obtained from preincubation with 100 /xl S9/plate. Therefore, 100 #l/plate $9 in the $9 mix was used as the microsomal activation system in the present studies. The numbers of revertants in the separate application of quercetin, AAF, 4NQO and B[a]P, and those in the mixed application of each AAF, 4NQO and B[a]P with quercetin are summarized in Table 1. 10/xg/plate of quercetin was mutagenic both with or without $9 mix in TA98 and TA100. However, the mutagenicity of quercetin was increased in the presence of $9 mix by as much as 3.5- and 4.7-fold in TA98 and TA100, respectively. Since AAF, 4NQO and B[a]P were also mutagenic, the numbers of revertants in the mixed application of AAF, 4NQO or B[a]P with quercetin were corn-

105 TABLE

1

MUTAGENICITY

OF AAF, 4NQO AND B[a]P WITH OR WITHOUT

Reagent

Querce-

Dose

Net r e v e r t a n t s / p l a t e a

tin b

(#g/plate)

TA98 - $9

Quercetin 2-Acetylamino- fluorene (AAF)

10

-Q +Q

-Q +Q

Benzo[a]pyrene (B[alP)

-Q +Q

a

TAI00 + $9

36+_ 5

268+_ 20

$9

+ $9

41+_ 8

193_+17

1

52+_ 11

5

282+_ 17

-

62-+ 9

688+_ 28

-

275+_36

2749 +- 128

-

687 + 89

1 + 10 5+10

4-Nitroquinoline-l-oxide (4NQO)

QUERCETIN

9+_ 6

0.01

23+_ 4

-

168+_ 17

0.1

252-+ 12

-

1405+_21

0.01 + 10

43+_ 2

-

115+_19

0.1 + 10

245+_14

-

1068+_39

1

-

69_+ 16

5

-

423 _+ 20

1 + 10

-

439-+ 32

-

295-+24

5 + 10

-

650+

-

440-+42

N u m b e r s o f s p o n t a n e o u s r e v e r t a n t s in D M S O ( T A 9 8 : - $ 9 ; t r a c t e d . D a t a s h o w the m e a n v a l u e _+ S . D , ( n = 9 ) .

30

24_+4, + $ 9 ; 5 9 + 9 , T A 1 0 0 : - $ 9 ;

-

80_+ 9 451 +_ 27

114_+6, + $ 9 ; 147_+ 15) w e r e s u b -

b - - Q ; A b s e n c e o f q u e r c e t i n , + Q ; P r e s e n c e o f 10 tzg/plate q u e r c e t i n .

pared with the sum of the numbers of revertants of AAF, 4NQO or B[a]P and revertants of quercetin in the separate application to check the enhancing or depressing effect of mutagenicity. The mutagenicity of the mixed application of AAF with quercetin was remarkably enhanced by as much as 2.2- and 5.0-fold in TA98 at doses of 1 and 5 tzg/plate of AAF, respectively, and 1.4- and 2.7-fold in TA100 in the presence of $9 mix. In the absence of $9 mix, the enhancement of mutagenicity was not observed in either TA98 or TA100. Quercetin did not enhance the mutagenicity of 4NQO with or without $9 mix, but rather decreased the mutagenicity of 4NQO in both strains. Mutagenicity of B[a]P (5 /~g/plate) with quercetin in TA100 was decreased (0.7-fold) in the presence of $9 mix. Enhancement of mutagenicity was observed in the mixed application of AAF with quercetin. However, whether AAF or quercetin has the comutagenic effect was not defined, because both AAF and quercetin are mutagenic in TA98 and

TA100. Dose-response curves for mutagenicity of quercetin with or without AAF were examined in the presence of $9 mix (Fig.2A and B). A linear dose-response curve of quercetin with 5 ~zg/plate AAF was observed in the range of 0-5 t~g/plate quercetin in spite of the absence of mutagenicity of quercetin alone in the same range in TA98 (Fig. 2A). Addition of quercetin (5 /~g/plate) usually enhanced the mutagenicity of AAF (8-fold). Therefore, these results suggest that the presence of quercetin up to levels equalling the molarity of AAF is effective in enhancing the mutagenicity of AAF. In TA100, similar results were observed, and the enhancement of the mutagenicity of AAF by addition of quercetin (5 t~g/plate) was about 10-fold. From these results, it seems that quercetin has a comutagenic effect on AAF. The mutation test using Salmonella typhimurium TA98/1,8 DNP6 was carried out to substantiate further that quercetin exerts a comutagenic effect on AAF. Table 2 shows the comparison of the number of revertants in TA98/1,8-DNP6 and TA98 in the

106

(A)

600

o 400

2000

o

i000

20O

5

i0 Quercetin

0

5

i0

(~g/plate)

Fig. 2. Effect of quercetin on mutagenicity of A A F in TA98 (A) and TAI00 (B). Mutagenicity of A A F (5 t~g/plate) with quercetin (e) and of quercetin only (©) were tested in the presence of $9 mix containing 20% of $9. Numbers of spontaneous revertants (TA98; 59-+ 9, TAI00; 147_+ 15) in DMSO with $9 mix were subtracted. Net revertants of 5 #g/plate A A F only were 282 _+ 17 and 62 _+9 in TA98 and TA100, respectively. Sum of revertants in the separate application of AAF (5 tzg/plate) and quercetin is also indicated (ix).

separate and the mixed application of AAF and quercetin with $9 mix. In the separate application, quercetin exerted the same activity in TA98/1,8-DNP6 as in TA98, which was also noted by McCoy et al. (1983) in the absence of $9 mix. The mutagenicity of AAF in TA98/1,8-DNP6 was reduced to 30°7o of its activity in TA98, since AAF is almost entirely dependent upon esterification to hydroxamic acid esters for expression of its

mutagenicity (McCoy et al., 1983). In the mixed application of AAF with quercetin in TA98/1,8 DNP6, mutagenic potencies of the mixture were also reduced to 55°7o and 45% of those in TA98 at doses of 1 and 5 /zg/plate AAF with 10 /~g/plate quercetin, respectively. However, the mutagenicity of mixed application of AAF with 10 #g/plate quercetin (380 revertants in 1 #g/plate of AAF and 1237 revertants in 5 #g/plate of AAF) were still enhanced by as much as 1.4- and 3.6-fold. These results indicate that the enhancement of mutagenicity in the mixed application is effective not only in TA98 but also in TA98/1,8-DNP6. Since a mutagenicity depressing effect of quercetin of B[a]P was observed in TA100 in the presence of $9 mix (Table 1), the dose-response curves for the mutagenicity of quercetin with or without 5 #g/plate B[a]P in the presence of $9 mix was examined in TA100 (Fig. 3). As can be seen in Fig. 3, the dose-response curve for mixed application of quercetin with 5 #g/plate B[a]P did not increase compared with that for quercetin alone. The mutagenicity of the mixed application of B[a]P with quercetin was remarkably reduced to about 60% of the sum of separate applications at doses ranging from 25 to 100 #g/plate of quercetin. These results suggest that quercetin has a desmutagenic effect on B[a]P. Considerable quantities of flavonoids including quercetin are consumed daily from foods (Brown, 1980); thus it is thought that the present results

TABLE 2 M U T A G E N I C I T Y OF A A F W I T H OR W I T H O U T Q U E R C E T I N IN T A 9 8 / I , 8 - D N P 6 A N D TA98 Reagent

Dose (#g/plate)

Net revertants/plate a TA98/1,8-DNP6

Quercetin 2-Acetylamino-fluorene (AAF)

A A F + Quercetin

a

10

257_+ 12

1 5

15 + 4 87+ 9

1 + 10 5 + 10

380+15 1237-+28

Ratio TA98/1,8-DNP6]

TA98 268_+ 20

0.96

52+ 282+

11 17

0.29 0.31

688+ 24 2749-+ 103

0.55 0.45

Numbers of spontaneous revertants in DMSO with $9 mix containing 20% of $9 (TA98; 59_+9, TA98/I,8-DNP6; 63 _+4) were subtracted. Results are the mean values +_ S.D. (n = 9).

107

thank Miss Yasuko Kanetsuna and Miss Mari Maeda for their excellent technical assistance in this investigation.

1200

....ax / /

800

t

t

/

t

f

References

.I i- - - ~ ' ~

f

/

4001

I

0

50 Quercetin

i00 (~g/plate)

Fig. 3. Effect of quercetin on mutagenicity of benzo[a]pyrene in TA100. The mutagenicity of B[a]P (5 gg/plate) with quercetin (O) and of quercetin only (O) were tested in the presence of $9 mix containing 20% of $9. Numbers of spontaneous revertants (139_+ 17) in DMSO with $9 mix were subtracted. Net revertants of 5 #g/plate B[a]P only was 451 + 27. Sum of net revertants in the separate application of B[a]P (5 #g/plate) and quercetin is also indicated (A). Each point is the mean value of 9 determinations.

may suggest the possibility of comutagenic and desmutagenic effects on other mutagens or carcinogens and stimulate new interest in the effects of flavonoids on human health. Acknowledgement The authors are grateful to Prof. B.N. Ames, University of California, Berkeley, who kindly supplied Salmonella typhimurium TA98 and TA100, and are also grateful to Prof. H.S. Rosenkranz, Case Western Reserve University, Cleveland, who kindly supplied Salmonella typhimurium TA98/1,8-DNP6 strains. We wish to

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. Bjeldanes, E.F., and G.W. Chang (1977) Mutagenic activity of quercetin and related compounds, Science, 197, 577-578. Boyland, E., E.R. Busby, C.E. Dukes, P.L. Grover and D. Manson (1964) Further experiments on implantation of materials into the urinary bladder of mice, Br. J. Cancer, 18, 575-581. Brown, J.P. (1980) A review of the genetic effects of naturally occuring flavonoids, anthraquinones and related compounds, Mutation Res., 75, 243-277. Brown, J.P., and P.S. Dietrich (1979) Mutagenicity of plant flavonoids in the Salmonella/mammalian microsome test: Activation of flavonol glycosides by mixed glycosidases from rat fecal bacteria and other sources, Mutation Res., 66, 223 -240. Herrmann, K. (1976) Flavonols and flavones in food plants: a review, J. Food Technol., 11, 433-448. Maron, D.M., and B.N. Ames (1983) Revised methods for the Salmonella mutagenicity test, Mutation Res., 113, 173-215. McCoy, E.C., M. Anders and H.S. Rosenkranz (1983) The basis of the insensitivity of Salmonella typhimurium strain TA98/I,8-DNP6 to the mutagenic action of nitroarenes, Mutation Res., 121, 17-23. Pamukcu, A.M., S. Yalaner, J.F. Hatcher and G.T. Bryan (1980) Quercetin, a rat intestinal and bladder carcinogen present in bracken fern (Pteridium aquilium), Cancer Res., 40, 3468-3471. Robinson, T. (1967) The organic constituents of higher plants, their chemistry and interrelationships, Burgess, Minneapolis, pp. 178-209. Sugimura, T., M. Nagao, T. Matsushima, S. Natori, K. Yoshihira, M. Fukuoka and M. Kuroyanagi (1977) Mutagenicity of flavone derivatives, Proc. Jpn. Acad., 53, Ser. B, 194-197. Yahagi, T., M. Nagao, Y. Seino, T. Matsushima, T. Sugimura and M. Okada (1977) Mutagenicities of N-nitrosamines on Salmonella, Mutation Res., 48, 121-130.