Mutation Research, 232 (1990) 243-248 Elsevier
243
MUT 04902
Metabolic activation of quercetin mutagenicity Raf Vrijsen 1, Yvette Michotte 2 and Albert Boey6 1 I Department of Microbiology and Hygiene and 2 Department of Pharmaceutical Chemistry, Vro'e Universiteit Brussel, B-1090 Brussels (Belgium) (Received 5 January 1990) (Revision received 28 March 1990) (Accepted 7 May 1990)
Keywords: Quercetin; Salmonella/microsome test; Metabolic activation
Summary The mutagenicity of quercetin was reinvestigated using the Salmonella/microsome test. The mutagenicity of quercetin was enhanced by the cytosolic fraction of liver extract (S100), or by ascorbate, and even more by the complete liver supernatant ($9) in the presence of cofactors (NADP and glucose-6-phosphate). The formation of metabolites by the $9 enzymes was demonstrated by reverse-phase HPLC.
Flavonoids are widely distributed in edible plants (Kiihnau, 1976). After it was reported that quercetin was mutagenic (Bjeldanes and Chang, 1977), more than 100 other flavonoids were examined using the Salmonella/microsome test. The flavonoids with mutagenic activity can be divided into 2 classes (MacGregor, 1984, 1986). The first class includes the flavonols (3-hydroxy flavones, e.g., quercetin and kaempferol) which are more mutagenic in tester strain TA98 than in TA100 (Brown and Dietrich, 1979; Hardigree and Epler, 1978), and the second the flavones (e.g., norwogonin) which are more mutagenic in strain TA100 than in strain TA98 (Nagao et al., 1981; Elliger et al., 1984). The first class can be further subdivided according to the requirements for metabolic activation. Quercetin, for instance, is weakly mutagenic even without metabolic activa-
Correspondence: Dr. R. Vrijsen, Department of Microbiology and Hygiene, Vrije Universiteit Brussel, Laarbeeklaan 103, B-1090 Brussels (Belgium).
tion, whereas kaempferol absolutely requires metabolic activation (MacGregor and Jurd, 1978; Brown and Dietrich, 1979). Concerning the mutagenicity of quercetin, Brown and Dietrich (loc. cit.) found that the enhancement of the quercetin mutagenicity by liver extracts was due to the cytosolic fraction (S100), and required neither cofactors nor induction of liver enzymes by Aroclor 1254. The cytosolic factors responsible for S100 enhancement were separated into 4 fractions by HPLC. One of these fractions was identified as superoxide dismutase. Since this enzyme protects quercetin from oxidative degradation, the enhancement of mutagenicity by S100 was ascribed to prevention of its degradation (Ochiai et al., 1984; Ueno et al., 1986). The results to be presented in this paper are in agreement with this interpretation. However, as will also be shown, the mutagenicity of quercetin is further increased by NADP- and glucose-6phosphate-dependent microsomal enzymes, indicating a contribution of metabolites. This conclusion was strengthened by the demonstration that
002%5107/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
244 quercetin metabolites were actually formed in the presence of the $9 supernatant. Materials and methods
Salmonella mutagenicity test The plate incorporation test was carried out essentially as described by Maron and Ames (1983) using tester strain TA98. To 2 ml molten top agar containing NaC1, histidine and biotin, 0.1 ml of an overnight culture ( + 108 bacteria per ml), 0.1 ml quercetin (Janssen Chimica; dissolved in ethanol and diluted in PBS, pH 7.2; the final amount of ethanol per plate was 50 /~1), and 0.5 ml $9 or S100 mix (see below) were added. The mixture was immediately poured onto Vogel-Bonner plates. The revertants were counted after 2 days' incubation at 37 o C. The following controls were included in every test: presence of the R-factor plasmid pKM101 (ampicillin resistance test using TA1538 as a control), rfa mutation (sensitivity for sodium deoxycholate), and number of spontaneous revertants.
cubated for 24 h at 37 o C and 765/~l 0.1 M H3PO 4 in methanol was added immediately after incubation. The samples were centrifuged at 12000 x g for 5 min to remove most of the protein. In preliminary experiments it was shown that quercetin was stable in this solution for at least 72 h. 20 /~I of each sample was used for HPLC analysis. The H P L C apparatus consisted of a Varian 2010 pump equipped with a Varian 2550 variable wavelength detector used at 370 nm and a D-2000 Chromato-Integrator (Merck-Hitachi). Separation was achieved on a Lichrocart RP-18 column (1 = 12.5 cm; i.d. 0.4 cm; particle size 5 /~m; Merck), using 45% 0.1 M phosphoric acid and 55% methanol as the mobile phase. Flow rate was 1 ml/min. In a preliminary experiment, quercetin was compared with luteolin and kaempferol, which differ from quercetin only by the absence of an OH group. The retention times for quercetin, luteolin and kaempferol were 11, 14 and 21 min, respectively (results not shown). Results
Preparation of liver extracts
Enhancement by liver homogenate fractions
The livers of 4 Sprague-Dawley rats were removed aseptically, washed twice in ice-cold 150 mM KC1, and minced in 3 volumes of 150 mM KC1. The livers were homogenized with a mixer (Ultra-Turrax, 45 s at 20000 rpm). The homogenate was centrifuged for 10 min at 9000 x g to obtain the $9 supernatant, and further for 1 h at 100000 x g for the $100 fraction. Both supernatants were kept in small portions at - 8 0 ° C. S9-I was similarly prepared from rats that were injected with 500 m g / k g Aroclor 1254 5 days before death.
As expected (Bjeldanes and Chang, 1977) quercetin by itself was mutagenic for Salmonella typhimurium strain TA98, and the number of revertants in excess of background increased linearly with the dose up to at least 120 /~g of quercetin per plate (Fig. 1). The mutagenicity was enhanced by addition of activation systems derived from rat liver, and the greatest enhancement was achieved with the $9 supernatant, supplemented with N A D P and glucose-6-phosphate. Proportionally, the greatest enhancement was observed at the lowest quercetin doses, and further studies were therefore conducted in the low range; however, the 15-#g dose represented the lower limit if the number of revertants was to stay well above the spontaneous background. A detailed study was conducted with 15/~g of quercetin per plate (Table 1, top half), supplemented with various liver homogenate fractions, with our without glucose-6-phosphate and NADP. By themselves, these cofactors did not significantly enhance quercetin mutagenicity. Three kinds of liver homogenate fractions were tested: the $9 supernatant containing the microsomes, the
Preparation of $9 and SIO0 mix To 3.5 or 7 ml liver extracts the following were added: 1 ml 0.4 M MgC12, 1.65 M KC1, 0.25 ml glucose-6-phosphate (0.8 M or 0.4 M), 2 ml N A D P (40 mM or 80 mM), 25 ml 0.2 M phosphate buffer, pH 7.4, and HzO q.s.p. 50 ml.
Reverse-phase HPLC To 600/~1 NaC1 (0.85%) and 150/zl $9 or $100 mix, 15/zl of an alcoholic quercetin stock solution (0.75 m g / m l ) was added. The samples were in-
245
W
3000
2000 z ¢
W
1000
1
I
I
0
40
80
I
120
Pg O.UERCETIN PER PLATE Fig. 1. Enhancement of quercetin mutagenicity by liver homogenate fractions, zx without liver homogenate; • 35/~1 S100 per plate; © S100 and cofactors (1 btmole glucose-6-phosphate and 0.8 /~mole N A D P ) per plate; • 35 /xl $9 per plate; [] $9 and cofactors per plate.
cytosolic S100 fraction of untreated animals, and the $9 fraction of Aroclor 1254-treated animals ($9-I). When the cofactors were omitted, all 3 kinds of homogenate fractions exerted the same,
roughly 3-fold (average 289% of basal level) enhancement of quercetin mutagenicity. The enhancement was slightly higher (average 407%) when S100 was used in combination with the cofactors. An even stronger enhancement (average 619% of basal level) was achieved by $9 (or $9-I) in combination with the cofactors. In summary, 3 levels of mutagenicity could be distinguished (the corresponding domains are separated by dashed lines in Table 1): (1) the basal level, which prevailed in the absence of liver extract, regardless of the addition of cofactors; (2) a medium level (grand average 336% of basal level), which was achieved with S100, or with $9 without cofactors; and (3) the top level (average 619%), which was achieved only with $9 (or $9-I) in combination with the cofactors. Though the differences were less pronounced with 30/~g of quercetin, the same 3 domains could still be distinguished (lower part of Table 1); in this case, the middle and top levels averaged 191% and 375% of the basal level, respectively. The experiment was repeated with liver extracts from a different set of animals, and yielded essentially the same results (not shown).
TABLE1 E N H A N C E M E N T O F M U T A G E N I C I T Y O F Q U E R C E T I N BY LIVER H O M O G E N A T E F R A C T I O N S A N D C O F A C T O R S Quercetin a
Cofactors a
Liver homogenate a
(/tg)
Glc6P
None
S100 35 ~1
$100 70 ~1
15
None
None
172 100
443 305
438 302
1.0
0.8
177 104
609 431
638 453
2.0
1.6
182 108
529 371
529 371
814 586
914 662
765 549
851 614
None
None
1.0
0.8
420 100 497 120
740 184 821 206
767 191 834 209
675 167 1364 348
730 182 1 659 426
721 179 1298 331
677 168 1334 341
2.0
1.6
481 116
805 201
894 225
1 478 379
1 687 434
1397 367
1473 377
30
NADP (t~ mole)
F
.
.
$9 35 ~1
$9 70 ~1
S9-1 35 ~1
$9-I 70 ~1
420 288
406 278
360 243
461 319
.
.
.
889 643
.
.
.
.
.
.
.
916 664
.
.
.
.
.
.
.
860 621
.
.
.
.
.
.
851 614
The actual number of revertants (average of 2 plates) is given in italics. From these figures, the n u m b e r of spontaneous revertants was subtracted (i.e., 40 per plate in this experiment, average of 18 plates without quercetin). The figures in the top and bottom half of the table were then normalized separately (roman characters), the basal level obtained with 15 or 30 #g quercetin, without liver homogenate or cofactors, being taken as 100 (boldfaced). A m o u n t per plate in a final volume of 2.7 ml; GIc6P: glucose-6-phosphate.
246
Equivalence of ascorbate and SlO0
ascorbate, the D and L isomers were equally active, and dehydroascorbate was inactive (results not shown). The top-level mutagenicity, achieved with $9 and cofactors, was not further enhanced by ascorbate (results not shown). Since the S100 fraction was equivalent to the antioxidant ascorbate with regard to quercetin mutagenicity, it is likely that the S100 effect was entirely due to protection of quercetin against oxidative degradation. Since all S100 components were also present in $9, the middle-level enhancement exerted by $9 without cofactors was probably due to the same effect. On the other hand, the top level of enhancement achieved by $9 with cofactors was presumably due to metabolic activation of quercetin.
Ochiai et al. (1984) identified the enzyme superoxide dismutase as a factor responsible for the enhancement of quercetin mutagenicity by the cytosolic S100 fraction, and ascribed the effect to stabilization of the structure of quercetin (we confirmed these findings; results not shown). Moreover, quercetin in aqueous solution is notoriously susceptible to oxidative degradation (Marklund and Marklund, 1974). Taken together, these facts suggested prevention of quercetin degradation as a possible explanation for the enhancement by the S100 fraction. If that explanation holds true, an antioxidant should have the same effect as the S100 fractions. To test this, the mutagenicity of quercetin was measured in the presence of increasing concentrations of L-ascorbate, as shown in Table 2. The effect of ascorbate was dose-dependent, levelling off at 120 /~g per plate. In confirmation of the hypothesis, there was a remarkable correlation between the enhancement by 120 /~g L-ascorbate and by S100 at each of the 3 quercetin doses. The distinction between a middle and top enhancement level, as defined in the previous section, was again evident. The middle level (average 451% of basal level) was reached either with 120 ~g Lascorbate or with S100, and the top level (average 967%) was achieved only with $9 and cofactors. In agreement with the antioxidant role ascribed to
Metabolization of quercetin To find out whether quercetin was actually metabolized by $9 with cofactors, reaction mixtures were prepared as for mutagenicity testing, omitting only the agar and test bacteria. The mixtures were incubated for 24 h at 37°C, deproteinated and analyzed by reverse-phase HPLC (see Materials and methods). In the absence of liver extracts with or without cofactors, quercetin after 24 h at 37 ° C was almost completely degraded (compare panels a and b in Fig. 2) and the remaining material absorbing at 370 nm was eluted near the solvent front. In the
TABLE 2 E N H A N C E M E N T OF M U T A G E N I C I T Y OF Q U E R C E T I N BY L-ASCORBATE Quercetin
a
L-Ascorbate a
Liver homogenate
None
15
144
290
340
411
416
510
1555
100
219
211
317
321
398
1247
227
613
892
1099
1317
1148
2180
100
287
423
523
629
547
1048
435
782
1113
1 572
1 768
1641
2528
100
184
264
375
422
391
606
30 60
15 ~g
30 /~g
60 #g
120 ~g
S100 35 ~tl
a
(/zg)
35 ~1 $ 9 + cofactors b
The actual number of revertants is given in italics. From these figures, the n u m b e r of spontaneous revertants was subtracted (i.e., 21 in this experiment, average count of 6 plates without quercetin). The figures for 15, 30 and 60 /Lg quercetin were normalized separately (roman characters), the basal level obtained with quercetin alone being taken as 100 (boldfaced). A m o u n t per plate in a final volume of 2.7 ml. b 1.0 ~mole ghicose-6-phosphate and 0.8/~mole N A D P per plate.
247
bl
Q
5
I
I
10
5
1'0
d
I 5
5
1'o
5
10
e
I 10
i
5 TIME
10 ( MIN )
Fig. 2. HPLC analysis of quercetin and metabolization products. Quercetin (7.35/~g in 1 ml sample solution; see Materials and methods) was chromatographed without prior incubation (a), or first incubated for 24 h at 37°C with the following admixtures: (b) cofactors (1 /~mole glucose-6-phosphate and 0.8/~mole NADH) only; (c) S100 (35 /d) only; (d) S100 and cofactors; (e) $9 (35 ~1) only; (f) $9 and cofactors.
presence of S100 with or without cofactors, the quercetin was not degraded, and there was little evidence of metabolization (panels c and d). When quercetin was incubated in the presence of $9 with or without cofactors, various metabolites were observed (panels e and f; it was verified that the 370-nm peaks were not due to absorption by the extract itself). The same 4 main peaks appeared with and without the cofactors, albeit in different proportions.
Discussion The present findings confirm that the S100 fraction of liver homogenate enhances quercetin mutagenicity, as originally shown by Brown and
Dietrich (1979); on the other hand, we disagree with these authors concerning the effect of the $9 enzymes. These authors observed only an insignificant enhancement by $9 and the cofactors. The failure to recognize the effect of $9 with cofactors may have been due to the accidental low score of the relevant petri dish in the only reported experiment, or to the use of a 30-/tg dose of quercetin. The extra enhancement given by $9 with cofactors is probably due to the formation of some potent mutagen(s) from quercetin. Our H P L C analyses confirm that $9, but not S100, caused the formation of a series of quercetin-derived products. However, with the exception of a minor peak, the same products were formed with and without the cofactors. Thus, the cofactors did not make much difference for the metabolization products of quercetin, whereas they were required to achieve the highest level of mutagenicity. The findings do not point directly to a given metabolite as the cause of the supplemental mutagenicity; but it remains possible that a minor metabolite is a much more potent mutagen than quercetin itself, like some metabolites of benzo[ a ]pyrene are much more mutagenic than the original compound (Wislocki et al., 1976a,b; Wood et al., 1976, 1977; Malaveille et al., 1977).
Acknowledgements The authors thank Annemiek Broos and Fabienne Smets for their excellent technical assistance, Bart Rombaut, Marc Detaevernier and J. Arany for their valuable advice, and Solange Peeters for help with the preparation of the manuscript.
References Bjeldanes, L.F., and G.W. Chang (1977) Mutagenic activity of quercetin and related compounds, Science, 197, 577-578. Brown, J.P., and P.S. Dietrich (1979) Mutagenicity of plant flavonols in the Salmonella/mammalian microsome test. Activation of flavonol glycosides by mixed glycosidases from rat cecal bacteria and other sources, Mutation Res., 66, 223-240. Elliger, C.A., P.R. Henika, and J.T. MacGregor (1984) Mutagenicity of flavones, chromones and acetophenones in Salmonella typhimurium. New structure-activity relationships, Mutation Res., 135, 77-86.
248 Hardigree, A.A., and J.L. Epler (1978) Comparative mutagenesis of plant flavonoids in microbial systems, Mutation Res., 58, 231-239. Ki~hnau, J. (1976) The flavonoids. A class of semi-essential food components: their role in human nutrition, Wld Rev. Nutr. Diet., 24, 117-191. MacGregor, J.T. (1984) Genetic and carcinogenic effects of plant flavonoids: an overview, Adv. Exp. Med. Biol., 177, 497-526. MacGregor, J.T. (1986) Mutagenic and carcinogenic effects of flavonoids, in: Plant Flavonoids in Biology and Medicine: Biochemical, Pharmacological, and Structure-Activity Relationships, A.R. Liss, New York, pp. 411-424. MacGregor, J.T., and L. Jurd (1978) Mutagenicity of plant flavonoids: structural requirements for mutagenic activity in Salmonella typhimurium, Mutation Res., 54, 297-309. Malaveille, C., T. Kuroki, P. Sims, P.L. Grover and H. Bartsch (1977) Mutagenicity of isomeric diol-epoxides of benzo[a]pyrene and benzo[a]anthracene in S. typhimurium TA98 and TA100 and in V79 Chinese hamster cells, Mutation Res., 44, 313-326. Marklund, S., and G. Marklund (1974) Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase, Eur. J. Biochem., 47, 469-474. Maron, D.M., and B.N. Ames (1983) Revised methods for the Salmonella mutagenicity test, Mutation Res., 113, 173-215. Nagao, M., N. Morita, T. Yahagi, M. Shimizu, M. Kuroyanagi, M. Fukuoka, K. Yoshihira, S. Natori, T. Fujino and T. Sugimura (1981) Mutagenicities of 61 flavonoids and 11 related compounds, Environ. Mutagen., 3, 401-419. Ochiai, M., M. Nagao, K. Wakabayashi and T. Sugimura
(1984) Superoxide dismutase acts as an enhancing factor for quercetin mutagenesis in rat-liver cytosol by preventing its decomposition, Mutation Res., 129, 19-24. Ueno, I., K. Haraikawa, M. Kohno, T. Hinomoto, H. OhyaNishiguchi, T. Tomatsuri and K. Yoshihira (1986) Possible involvement of superoxide dismutase in the mutagenicity of quercetin in Salmonella typhimurium strain TA98, in: Plant Flavonoids in Biology and Medicine: Biochemical, Pharmacological, and Structure-Activity relationships, A.R. Liss, New York, pp. 425-428. Wislocki, P.G., A.W. Wood, R.L. Chang, W. Levin, H. Yagi, O. Hernandez, P.M. Dansette, D.M. Jerina and A.H. Conney (1976a) Mutagenicity and cytotoxicity of benzo[a]pyrene, arene oxides, phenols, quinones, and dihydrodiols in bacterial and mammalian cells, Cancer Res., 36, 33503357. Wislocki, P.G., A.W. Wood, R.L. Chang, W. Levin, H. Yagi, O. Hernandez, D.M. Jerina and A.H. Conney (1976b) High mutagenicity and toxicity of a diol epoxide derived from benzo[a]pyrene, Biochem. Biophys. Res. Commun., 68, 1006-1012. Wood, A.W., P.G. Wislocki, R.L. Chang, W. Levin, A.Y.H. Lu, H. Yagi, O. Hernandez, D.M. Jerina and A.H. Conney (1976) Mutagenicity and cytotoxicity of benzo[a]pyrene benzo-ring epoxides, Cancer Res., 36, 3358-3366. Wood, A.W., R.L. Chang, W. Levin, Y. Yagi, D.R. Thakker, D.M. Jerina and A.H. Conney (1977) Differences in mutagenicity of the optical enantiomers of the diastereomeric benzo[a]pyrene 7,8-diol-9,10-epoxides, Biochem. Biophys. Res. Commun., 77, 1389-1396.
243
MUT 04902
Metabolic activation of quercetin mutagenicity Raf Vrijsen 1, Yvette Michotte 2 and Albert Boey6 1 I Department of Microbiology and Hygiene and 2 Department of Pharmaceutical Chemistry, Vro'e Universiteit Brussel, B-1090 Brussels (Belgium) (Received 5 January 1990) (Revision received 28 March 1990) (Accepted 7 May 1990)
Keywords: Quercetin; Salmonella/microsome test; Metabolic activation
Summary The mutagenicity of quercetin was reinvestigated using the Salmonella/microsome test. The mutagenicity of quercetin was enhanced by the cytosolic fraction of liver extract (S100), or by ascorbate, and even more by the complete liver supernatant ($9) in the presence of cofactors (NADP and glucose-6-phosphate). The formation of metabolites by the $9 enzymes was demonstrated by reverse-phase HPLC.
Flavonoids are widely distributed in edible plants (Kiihnau, 1976). After it was reported that quercetin was mutagenic (Bjeldanes and Chang, 1977), more than 100 other flavonoids were examined using the Salmonella/microsome test. The flavonoids with mutagenic activity can be divided into 2 classes (MacGregor, 1984, 1986). The first class includes the flavonols (3-hydroxy flavones, e.g., quercetin and kaempferol) which are more mutagenic in tester strain TA98 than in TA100 (Brown and Dietrich, 1979; Hardigree and Epler, 1978), and the second the flavones (e.g., norwogonin) which are more mutagenic in strain TA100 than in strain TA98 (Nagao et al., 1981; Elliger et al., 1984). The first class can be further subdivided according to the requirements for metabolic activation. Quercetin, for instance, is weakly mutagenic even without metabolic activa-
Correspondence: Dr. R. Vrijsen, Department of Microbiology and Hygiene, Vrije Universiteit Brussel, Laarbeeklaan 103, B-1090 Brussels (Belgium).
tion, whereas kaempferol absolutely requires metabolic activation (MacGregor and Jurd, 1978; Brown and Dietrich, 1979). Concerning the mutagenicity of quercetin, Brown and Dietrich (loc. cit.) found that the enhancement of the quercetin mutagenicity by liver extracts was due to the cytosolic fraction (S100), and required neither cofactors nor induction of liver enzymes by Aroclor 1254. The cytosolic factors responsible for S100 enhancement were separated into 4 fractions by HPLC. One of these fractions was identified as superoxide dismutase. Since this enzyme protects quercetin from oxidative degradation, the enhancement of mutagenicity by S100 was ascribed to prevention of its degradation (Ochiai et al., 1984; Ueno et al., 1986). The results to be presented in this paper are in agreement with this interpretation. However, as will also be shown, the mutagenicity of quercetin is further increased by NADP- and glucose-6phosphate-dependent microsomal enzymes, indicating a contribution of metabolites. This conclusion was strengthened by the demonstration that
002%5107/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
244 quercetin metabolites were actually formed in the presence of the $9 supernatant. Materials and methods
Salmonella mutagenicity test The plate incorporation test was carried out essentially as described by Maron and Ames (1983) using tester strain TA98. To 2 ml molten top agar containing NaC1, histidine and biotin, 0.1 ml of an overnight culture ( + 108 bacteria per ml), 0.1 ml quercetin (Janssen Chimica; dissolved in ethanol and diluted in PBS, pH 7.2; the final amount of ethanol per plate was 50 /~1), and 0.5 ml $9 or S100 mix (see below) were added. The mixture was immediately poured onto Vogel-Bonner plates. The revertants were counted after 2 days' incubation at 37 o C. The following controls were included in every test: presence of the R-factor plasmid pKM101 (ampicillin resistance test using TA1538 as a control), rfa mutation (sensitivity for sodium deoxycholate), and number of spontaneous revertants.
cubated for 24 h at 37 o C and 765/~l 0.1 M H3PO 4 in methanol was added immediately after incubation. The samples were centrifuged at 12000 x g for 5 min to remove most of the protein. In preliminary experiments it was shown that quercetin was stable in this solution for at least 72 h. 20 /~I of each sample was used for HPLC analysis. The H P L C apparatus consisted of a Varian 2010 pump equipped with a Varian 2550 variable wavelength detector used at 370 nm and a D-2000 Chromato-Integrator (Merck-Hitachi). Separation was achieved on a Lichrocart RP-18 column (1 = 12.5 cm; i.d. 0.4 cm; particle size 5 /~m; Merck), using 45% 0.1 M phosphoric acid and 55% methanol as the mobile phase. Flow rate was 1 ml/min. In a preliminary experiment, quercetin was compared with luteolin and kaempferol, which differ from quercetin only by the absence of an OH group. The retention times for quercetin, luteolin and kaempferol were 11, 14 and 21 min, respectively (results not shown). Results
Preparation of liver extracts
Enhancement by liver homogenate fractions
The livers of 4 Sprague-Dawley rats were removed aseptically, washed twice in ice-cold 150 mM KC1, and minced in 3 volumes of 150 mM KC1. The livers were homogenized with a mixer (Ultra-Turrax, 45 s at 20000 rpm). The homogenate was centrifuged for 10 min at 9000 x g to obtain the $9 supernatant, and further for 1 h at 100000 x g for the $100 fraction. Both supernatants were kept in small portions at - 8 0 ° C. S9-I was similarly prepared from rats that were injected with 500 m g / k g Aroclor 1254 5 days before death.
As expected (Bjeldanes and Chang, 1977) quercetin by itself was mutagenic for Salmonella typhimurium strain TA98, and the number of revertants in excess of background increased linearly with the dose up to at least 120 /~g of quercetin per plate (Fig. 1). The mutagenicity was enhanced by addition of activation systems derived from rat liver, and the greatest enhancement was achieved with the $9 supernatant, supplemented with N A D P and glucose-6-phosphate. Proportionally, the greatest enhancement was observed at the lowest quercetin doses, and further studies were therefore conducted in the low range; however, the 15-#g dose represented the lower limit if the number of revertants was to stay well above the spontaneous background. A detailed study was conducted with 15/~g of quercetin per plate (Table 1, top half), supplemented with various liver homogenate fractions, with our without glucose-6-phosphate and NADP. By themselves, these cofactors did not significantly enhance quercetin mutagenicity. Three kinds of liver homogenate fractions were tested: the $9 supernatant containing the microsomes, the
Preparation of $9 and SIO0 mix To 3.5 or 7 ml liver extracts the following were added: 1 ml 0.4 M MgC12, 1.65 M KC1, 0.25 ml glucose-6-phosphate (0.8 M or 0.4 M), 2 ml N A D P (40 mM or 80 mM), 25 ml 0.2 M phosphate buffer, pH 7.4, and HzO q.s.p. 50 ml.
Reverse-phase HPLC To 600/~1 NaC1 (0.85%) and 150/zl $9 or $100 mix, 15/zl of an alcoholic quercetin stock solution (0.75 m g / m l ) was added. The samples were in-
245
W
3000
2000 z ¢
W
1000
1
I
I
0
40
80
I
120
Pg O.UERCETIN PER PLATE Fig. 1. Enhancement of quercetin mutagenicity by liver homogenate fractions, zx without liver homogenate; • 35/~1 S100 per plate; © S100 and cofactors (1 btmole glucose-6-phosphate and 0.8 /~mole N A D P ) per plate; • 35 /xl $9 per plate; [] $9 and cofactors per plate.
cytosolic S100 fraction of untreated animals, and the $9 fraction of Aroclor 1254-treated animals ($9-I). When the cofactors were omitted, all 3 kinds of homogenate fractions exerted the same,
roughly 3-fold (average 289% of basal level) enhancement of quercetin mutagenicity. The enhancement was slightly higher (average 407%) when S100 was used in combination with the cofactors. An even stronger enhancement (average 619% of basal level) was achieved by $9 (or $9-I) in combination with the cofactors. In summary, 3 levels of mutagenicity could be distinguished (the corresponding domains are separated by dashed lines in Table 1): (1) the basal level, which prevailed in the absence of liver extract, regardless of the addition of cofactors; (2) a medium level (grand average 336% of basal level), which was achieved with S100, or with $9 without cofactors; and (3) the top level (average 619%), which was achieved only with $9 (or $9-I) in combination with the cofactors. Though the differences were less pronounced with 30/~g of quercetin, the same 3 domains could still be distinguished (lower part of Table 1); in this case, the middle and top levels averaged 191% and 375% of the basal level, respectively. The experiment was repeated with liver extracts from a different set of animals, and yielded essentially the same results (not shown).
TABLE1 E N H A N C E M E N T O F M U T A G E N I C I T Y O F Q U E R C E T I N BY LIVER H O M O G E N A T E F R A C T I O N S A N D C O F A C T O R S Quercetin a
Cofactors a
Liver homogenate a
(/tg)
Glc6P
None
S100 35 ~1
$100 70 ~1
15
None
None
172 100
443 305
438 302
1.0
0.8
177 104
609 431
638 453
2.0
1.6
182 108
529 371
529 371
814 586
914 662
765 549
851 614
None
None
1.0
0.8
420 100 497 120
740 184 821 206
767 191 834 209
675 167 1364 348
730 182 1 659 426
721 179 1298 331
677 168 1334 341
2.0
1.6
481 116
805 201
894 225
1 478 379
1 687 434
1397 367
1473 377
30
NADP (t~ mole)
F
.
.
$9 35 ~1
$9 70 ~1
S9-1 35 ~1
$9-I 70 ~1
420 288
406 278
360 243
461 319
.
.
.
889 643
.
.
.
.
.
.
.
916 664
.
.
.
.
.
.
.
860 621
.
.
.
.
.
.
851 614
The actual number of revertants (average of 2 plates) is given in italics. From these figures, the n u m b e r of spontaneous revertants was subtracted (i.e., 40 per plate in this experiment, average of 18 plates without quercetin). The figures in the top and bottom half of the table were then normalized separately (roman characters), the basal level obtained with 15 or 30 #g quercetin, without liver homogenate or cofactors, being taken as 100 (boldfaced). A m o u n t per plate in a final volume of 2.7 ml; GIc6P: glucose-6-phosphate.
246
Equivalence of ascorbate and SlO0
ascorbate, the D and L isomers were equally active, and dehydroascorbate was inactive (results not shown). The top-level mutagenicity, achieved with $9 and cofactors, was not further enhanced by ascorbate (results not shown). Since the S100 fraction was equivalent to the antioxidant ascorbate with regard to quercetin mutagenicity, it is likely that the S100 effect was entirely due to protection of quercetin against oxidative degradation. Since all S100 components were also present in $9, the middle-level enhancement exerted by $9 without cofactors was probably due to the same effect. On the other hand, the top level of enhancement achieved by $9 with cofactors was presumably due to metabolic activation of quercetin.
Ochiai et al. (1984) identified the enzyme superoxide dismutase as a factor responsible for the enhancement of quercetin mutagenicity by the cytosolic S100 fraction, and ascribed the effect to stabilization of the structure of quercetin (we confirmed these findings; results not shown). Moreover, quercetin in aqueous solution is notoriously susceptible to oxidative degradation (Marklund and Marklund, 1974). Taken together, these facts suggested prevention of quercetin degradation as a possible explanation for the enhancement by the S100 fraction. If that explanation holds true, an antioxidant should have the same effect as the S100 fractions. To test this, the mutagenicity of quercetin was measured in the presence of increasing concentrations of L-ascorbate, as shown in Table 2. The effect of ascorbate was dose-dependent, levelling off at 120 /~g per plate. In confirmation of the hypothesis, there was a remarkable correlation between the enhancement by 120 /~g L-ascorbate and by S100 at each of the 3 quercetin doses. The distinction between a middle and top enhancement level, as defined in the previous section, was again evident. The middle level (average 451% of basal level) was reached either with 120 ~g Lascorbate or with S100, and the top level (average 967%) was achieved only with $9 and cofactors. In agreement with the antioxidant role ascribed to
Metabolization of quercetin To find out whether quercetin was actually metabolized by $9 with cofactors, reaction mixtures were prepared as for mutagenicity testing, omitting only the agar and test bacteria. The mixtures were incubated for 24 h at 37°C, deproteinated and analyzed by reverse-phase HPLC (see Materials and methods). In the absence of liver extracts with or without cofactors, quercetin after 24 h at 37 ° C was almost completely degraded (compare panels a and b in Fig. 2) and the remaining material absorbing at 370 nm was eluted near the solvent front. In the
TABLE 2 E N H A N C E M E N T OF M U T A G E N I C I T Y OF Q U E R C E T I N BY L-ASCORBATE Quercetin
a
L-Ascorbate a
Liver homogenate
None
15
144
290
340
411
416
510
1555
100
219
211
317
321
398
1247
227
613
892
1099
1317
1148
2180
100
287
423
523
629
547
1048
435
782
1113
1 572
1 768
1641
2528
100
184
264
375
422
391
606
30 60
15 ~g
30 /~g
60 #g
120 ~g
S100 35 ~tl
a
(/zg)
35 ~1 $ 9 + cofactors b
The actual number of revertants is given in italics. From these figures, the n u m b e r of spontaneous revertants was subtracted (i.e., 21 in this experiment, average count of 6 plates without quercetin). The figures for 15, 30 and 60 /Lg quercetin were normalized separately (roman characters), the basal level obtained with quercetin alone being taken as 100 (boldfaced). A m o u n t per plate in a final volume of 2.7 ml. b 1.0 ~mole ghicose-6-phosphate and 0.8/~mole N A D P per plate.
247
bl
Q
5
I
I
10
5
1'0
d
I 5
5
1'o
5
10
e
I 10
i
5 TIME
10 ( MIN )
Fig. 2. HPLC analysis of quercetin and metabolization products. Quercetin (7.35/~g in 1 ml sample solution; see Materials and methods) was chromatographed without prior incubation (a), or first incubated for 24 h at 37°C with the following admixtures: (b) cofactors (1 /~mole glucose-6-phosphate and 0.8/~mole NADH) only; (c) S100 (35 /d) only; (d) S100 and cofactors; (e) $9 (35 ~1) only; (f) $9 and cofactors.
presence of S100 with or without cofactors, the quercetin was not degraded, and there was little evidence of metabolization (panels c and d). When quercetin was incubated in the presence of $9 with or without cofactors, various metabolites were observed (panels e and f; it was verified that the 370-nm peaks were not due to absorption by the extract itself). The same 4 main peaks appeared with and without the cofactors, albeit in different proportions.
Discussion The present findings confirm that the S100 fraction of liver homogenate enhances quercetin mutagenicity, as originally shown by Brown and
Dietrich (1979); on the other hand, we disagree with these authors concerning the effect of the $9 enzymes. These authors observed only an insignificant enhancement by $9 and the cofactors. The failure to recognize the effect of $9 with cofactors may have been due to the accidental low score of the relevant petri dish in the only reported experiment, or to the use of a 30-/tg dose of quercetin. The extra enhancement given by $9 with cofactors is probably due to the formation of some potent mutagen(s) from quercetin. Our H P L C analyses confirm that $9, but not S100, caused the formation of a series of quercetin-derived products. However, with the exception of a minor peak, the same products were formed with and without the cofactors. Thus, the cofactors did not make much difference for the metabolization products of quercetin, whereas they were required to achieve the highest level of mutagenicity. The findings do not point directly to a given metabolite as the cause of the supplemental mutagenicity; but it remains possible that a minor metabolite is a much more potent mutagen than quercetin itself, like some metabolites of benzo[ a ]pyrene are much more mutagenic than the original compound (Wislocki et al., 1976a,b; Wood et al., 1976, 1977; Malaveille et al., 1977).
Acknowledgements The authors thank Annemiek Broos and Fabienne Smets for their excellent technical assistance, Bart Rombaut, Marc Detaevernier and J. Arany for their valuable advice, and Solange Peeters for help with the preparation of the manuscript.
References Bjeldanes, L.F., and G.W. Chang (1977) Mutagenic activity of quercetin and related compounds, Science, 197, 577-578. Brown, J.P., and P.S. Dietrich (1979) Mutagenicity of plant flavonols in the Salmonella/mammalian microsome test. Activation of flavonol glycosides by mixed glycosidases from rat cecal bacteria and other sources, Mutation Res., 66, 223-240. Elliger, C.A., P.R. Henika, and J.T. MacGregor (1984) Mutagenicity of flavones, chromones and acetophenones in Salmonella typhimurium. New structure-activity relationships, Mutation Res., 135, 77-86.
248 Hardigree, A.A., and J.L. Epler (1978) Comparative mutagenesis of plant flavonoids in microbial systems, Mutation Res., 58, 231-239. Ki~hnau, J. (1976) The flavonoids. A class of semi-essential food components: their role in human nutrition, Wld Rev. Nutr. Diet., 24, 117-191. MacGregor, J.T. (1984) Genetic and carcinogenic effects of plant flavonoids: an overview, Adv. Exp. Med. Biol., 177, 497-526. MacGregor, J.T. (1986) Mutagenic and carcinogenic effects of flavonoids, in: Plant Flavonoids in Biology and Medicine: Biochemical, Pharmacological, and Structure-Activity Relationships, A.R. Liss, New York, pp. 411-424. MacGregor, J.T., and L. Jurd (1978) Mutagenicity of plant flavonoids: structural requirements for mutagenic activity in Salmonella typhimurium, Mutation Res., 54, 297-309. Malaveille, C., T. Kuroki, P. Sims, P.L. Grover and H. Bartsch (1977) Mutagenicity of isomeric diol-epoxides of benzo[a]pyrene and benzo[a]anthracene in S. typhimurium TA98 and TA100 and in V79 Chinese hamster cells, Mutation Res., 44, 313-326. Marklund, S., and G. Marklund (1974) Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase, Eur. J. Biochem., 47, 469-474. Maron, D.M., and B.N. Ames (1983) Revised methods for the Salmonella mutagenicity test, Mutation Res., 113, 173-215. Nagao, M., N. Morita, T. Yahagi, M. Shimizu, M. Kuroyanagi, M. Fukuoka, K. Yoshihira, S. Natori, T. Fujino and T. Sugimura (1981) Mutagenicities of 61 flavonoids and 11 related compounds, Environ. Mutagen., 3, 401-419. Ochiai, M., M. Nagao, K. Wakabayashi and T. Sugimura
(1984) Superoxide dismutase acts as an enhancing factor for quercetin mutagenesis in rat-liver cytosol by preventing its decomposition, Mutation Res., 129, 19-24. Ueno, I., K. Haraikawa, M. Kohno, T. Hinomoto, H. OhyaNishiguchi, T. Tomatsuri and K. Yoshihira (1986) Possible involvement of superoxide dismutase in the mutagenicity of quercetin in Salmonella typhimurium strain TA98, in: Plant Flavonoids in Biology and Medicine: Biochemical, Pharmacological, and Structure-Activity relationships, A.R. Liss, New York, pp. 425-428. Wislocki, P.G., A.W. Wood, R.L. Chang, W. Levin, H. Yagi, O. Hernandez, P.M. Dansette, D.M. Jerina and A.H. Conney (1976a) Mutagenicity and cytotoxicity of benzo[a]pyrene, arene oxides, phenols, quinones, and dihydrodiols in bacterial and mammalian cells, Cancer Res., 36, 33503357. Wislocki, P.G., A.W. Wood, R.L. Chang, W. Levin, H. Yagi, O. Hernandez, D.M. Jerina and A.H. Conney (1976b) High mutagenicity and toxicity of a diol epoxide derived from benzo[a]pyrene, Biochem. Biophys. Res. Commun., 68, 1006-1012. Wood, A.W., P.G. Wislocki, R.L. Chang, W. Levin, A.Y.H. Lu, H. Yagi, O. Hernandez, D.M. Jerina and A.H. Conney (1976) Mutagenicity and cytotoxicity of benzo[a]pyrene benzo-ring epoxides, Cancer Res., 36, 3358-3366. Wood, A.W., R.L. Chang, W. Levin, Y. Yagi, D.R. Thakker, D.M. Jerina and A.H. Conney (1977) Differences in mutagenicity of the optical enantiomers of the diastereomeric benzo[a]pyrene 7,8-diol-9,10-epoxides, Biochem. Biophys. Res. Commun., 77, 1389-1396.
