Mutation Research, 265 (1992) 75-81
75
© 1992 Elsevier Science Publishers B.V. All rights reserved 0027-5107/92/$05.00
MUT 05035
Oxygen species and the genotoxicity of quercetin Jose Rueff 1, Ant6nio Laires 1,2,Jorge Gaspar l, Helena Borba 1 and Ant6nio Rodrigues 1 1 Department of Genetics, Faculty of Medical Sciences UNL, Lisbon and 2 Faculty of Sciences and Technology UNL, Monte da Caparica (Portugal)
(Received 3 April 1991) (Revision received 10 July 1991) (Accepted 22 July 1991)
Keywords: Quercetin; Oxygen species; SOS induction
Summary Quercetin has been extensively studied in various short-term assays for genotoxicity. The patterns of genotoxicity of quercetin for different genetic endpoints are subject to a variety of factors (pH, antioxidants, metabolism) whose precise role in each test remains unclear. In the present study we report on the possible effect of oxygen-derived species on the activity of quercetin in the Ames assay and in the SOS chromotest. Our results seem to suggest that superoxide dismutase (SOD) does not account for the levels of mutagenicity detected in the presence of $9 or S100. The latter may, however, contain other factors of antioxidant defense which may prevent the oxidative degradation of quercetin. Since this degradation occurs at pH values above neutrality and the SOS-inducing activity is higher at pH 6.0, it is concluded that the response of quercetin in the SOS chromotest is due to quercetin itself at acidic pH. The SOS-inducing activity at pH 7.4 is enhanced by SOD, but it cannot be unambiguously concluded that this effect in the SOS chromotest might only be due to protection against the oxidative degradation of quercetin.
D N A damage by quercetin may occur via more than one mechanism. Reports have appeared on the influence of the mammalian microsome system ($9) on the activity of quercetin towards various genetic end-points. The results obtained using the Ames assay, the induction of sisterchromatid exchanges in human lymphocytes and
Correspondence: Dr. J. Rueff, Department of Genetics, Faculty of Medical Sciences UNL, R. Junqueira 96, P-1300 Lisbon (Portugal).
the induction of some of the genes of the SOS system in E. coli (suM, recA and u m u C ) seem to point to the conclusion that quercetin exerts its genotoxic activity either directly or through putative degradation products depending on the genetic end-point assessed (Rueff et al., 1986, 1989; Llagostera et al., 1987). In the L5178Y TK +/- system (Meltz and MacGregor, 1981) the mutagenicity of quercetin has been shown to be lowered by the presence of $9, and in C H O cells quercetin was likewise genotoxic only in the' absence of $9 (Carver et al., 1983).
76
In the Ames assay, however, the mutagenicity of quercetin is consistently enhanced by the presence of $9 (Bjeldanes and Chang, 1977; MacGregor and Jurd, 1978; Brown and Dietrich, 1979; Hatcher and Bryan, 1985; Rueff et al., 1986; Vrijsen et al., 1990). The enhancement of the mutagenicity of quercetin in the Ames assay has been shown to depend on the cytosolic (S100) fraction present in $9 rather than on microsomal enzymes (Brown and Dietrich, 1979; Ochiai et al., 1984; Rueff et al., 1986). The possible metabolite(s) of quercetin which might be responsible for the enhancement of mutagenicity in the Ames assay still await identification. Yet, quercetin spontaneously gives rise to the superoxide anion as a function of increasing pH values and is degraded in this autooxidative process with formation of a product detectable by HPLC (Ueno et al., 1984; Rueff et al., 1989). These observations may lead to 2 preliminary hypotheses: (i) in the Ames assay the mutagenicity of quercetin is enhanced due to protection afforded by the copper-zinc superoxide dismutase (SOD) present in $9 (Ochiai et al., 1984); (ii) in other assay systems, e.g., SOS chromotest, sister-chromatid exchanges, chromosomal aberrations, the genotoxicity might be due instead to oxygen-derived species produced by spontaneous degradation of quercetin, which would explain the decreased response in the presence of $9 (Carver et al., 1983; Rueff et al., 1986). Vrijsen et al. (1990) have recently reported that $9, but not S100, is capable of metabolizing quercetin which could help in explaining the enhancement of activity in the Ames assay. Along this line we report here on additional studies performed to address the hypotheses put forward above.
contents in the $9 and S100 fractions were determined according to Lowry et al. (1951). Enzyme assays Superoxide dismutase (SOD) activities in $9 and S100 were determined as described by Marklund and Marklund (1974). One enzyme unit is the amount of protein that inhibits the autooxidation of pyrogallol at pH 8.0 by 50%, followed at 420 nm. Catalase (CAT) activities in $9 and S100 were determined as described by Sinha (1972). The method is based on the reduction of potassium dichromate in acetic acid to chromic acetate when heated in the presence of hydrogen peroxide, measured at 570 nm. The catalase contents of $9 and S100 were expressed in terms of rate constant (Kat) per g of protein. Cytochrome P450 contents of $9 and S100 were determined from CO difference spectra of dithionite-reduced samples of $9 and S100 according to Omura and Sato (1964).
Materials and methods
Ames test Salmonella typhimurium TA98, obtained from Professor B.N. Ames (Berkeley, CA), was used as tester strain. Mutagenicity testing in the Ames assay was carried out as previously described (Maron and Ames, 1983; Rueff et al., 1986). For the assays carried out in the presence of SOD (EC 1.1.5.1.1) and catalase (EC 1.11.1.6), both from Sigma, the enzymes were prepared, respectively, in phosphate buffer 0.1 M, pH 7.4 and 0.02 M, pH 7.4. Diagnostic mutagens were 2-nitrofluorene (4.7 nmole/plate without $9) and benzo[a]pyrene (19.8 nmole/plate). At least 2 independent experiments were carried out for each dose level. Mutagenic activity was determined from the slope of the least squares line of the linear portion of the dose-response curve.
Rat liver enzymes $9 was prepared as described previously (Laires et al., 1982; Maron and Ames, 1983). The S100 fraction was prepared by centrifuging fresh $9 for 1 h at 100,000 x g. The supernatants were used routinely in $9 mix and S100 mix, without and with cofactors (glucose-6-phosphate and NADP) at a level of 0.1 ml per 1.0 ml of mix. The protein
SOS chromotest Induction of SOS functions was assayed with the SOS chromotest by determining the induced levels of fl-galactosidase whose gene, lacZ, had been placed under the control of sulA, one of the SOS genes involved in cell division inhibition (Quillardet and Hofnung, 1985). The strain Escherichia coli PQ37 was obtained from Professor
77
M. Hofnung (Paris). The SOS chomotest was performed as described by Quillardet and Hofnung (1985) with the adaptation introduced by Marzin et al. (1986). This consisted of kinetic measurements of /3-galactosidase and alkaline phosphatase activities instead of 'end-point' assays. When testing the SOS-inducing activity of quercetin at pH 6.0 or pH 7.4 the dilution 1 : 10 of the culture was made in LB/phosphate buffer, pH 6.0 or pH 7.4, 1:1, respectively. For the assays carried out in the presence of SOD (9.1 Marklund units per assay) the enzyme was prepared in 0.2 M phosphate buffers, pH 6.0 and pH 7.4. For the assays carried out in the presence of catalase (14 units per assay), the enzyme was prepared in 0.02 M phosphate buffer, pH 7.4. The diagnostic mutagen was 4-nitroquinoline-Noxide (1.05 nmole/assay). At least 2 independent experiments were carried out for each dose level. Results
The catalase and superoxide dismutase activities in $9 and S100 used in the present study are presented in Table 1 along with the cytochrome P450 content. The experiments carried out to ascertain the role of catalase and superoxide dismutase on the mutagenic activity of 33 nmole quercetin in strain TA98 are presented in Table 2 along with the response in the presence of $9 and S100. Catalase has no effect on the mutagenicity of
TABLE 1 E N Z Y M A T I C ACTIVITIES O F S U P E R O X 1 D E DISMUT A S E (SOD), C A T A L A S E (CAT) A N D Cyt P450 1N $9 A N D S100 $9 SOD ( u n i t s / m g protein) CAT ( K a t / g protein) Cyt P450 ( n m o l e / m g protein)
S100 7.1
22.0
292
4211
0.77
n.d.
n.d., not detectable.
quercetin. SOD at a level higher than present in S100 or $9 (30 Marklund units vs. 22 in S100 and 13 in $9) exerts an enhancing effect on the mutagenicity of quercetin albeit lower than that observable with $9 or S100. The superoxide anion does not respond in strain TA98 as shown in Table 2 using the xanthine/xanthine oxidase system. The absence of response of superoxide is in agreement with the results of Ueno et al. (1984) and De Flora et al. (1989). The xanthine/ xanthine oxidase system completely abolishes the mutagenicity of quercetin through degradation by superoxide (Table 2). Dose-response curves of quercetin in the presence of 13 units SOD, and in the presence of $9 mix, S100 mix and these 2 extracts with added cofactors are presented in Fig. 1.
TABLE 2 R O L E O F S U P E R O X 1 D E D I S M U T A S E (SOD), C A T A L A S E (CAT) A N D X A N T H 1 N E / X A N T H I N E O X I D A S E ( X / X O ) ON T H E M U T A G E N I C I T Y O F Q U E R C E T I N ~' Control Quercetin (0 nmole) Quercetin (33 nmole)
$9 mix b (500 ~1)
S100 mix c (500 p.I)
SOD (30 U) 0
CAT (100 U) ~
X/XO r
29
46
27
25
31
35
120
276
247
164
127
30
R e v . / p l a t e in strain TA98. Three independent experiments. b 13 S O D Marklund u n i t s / 5 0 0 ~ l $9 mix. c 22 S O D Marklund u n i t s / 5 0 0 ~ l $100 mix. d Marklund units. O n e unit decomposes 1 /~mole of H 2 0 2 / m i n at pH 7.0 and 2 5 ° C . f X = 15 ~ g / p l a t e ; XO = 0.0021 u n i t s / p l a t e .
78
Statistical analysis of the regression output (F-test, multiple comparison of Scheff6) shows that quercetin mutagenicity is not significantly increased in the presence of SOD. Thus, SOD at levels higher than present in $9 does not afford enough protection against the autooxidative degradation of quercetin. Both $9 mix and S100 mix significantly enhanced quercetin mutagenicity ( p < 0.05). When $9 mix is prepared without the cofactors needed for cytochrome P450-mediated metabolism the response is significantly lower ( p < 0.05). The absence of cofactors in $100 mix, however, does not result in any statistically significant effect as compared with S100 with cofactors. Both $9 mix and S100 mix in the absence of cofactors exert the same enhancing effect on the mutagenicity of quercetin. As shown in Fig. 2 the SOS-inducing activity of quercetin is significantly higher at p H 6.0 as compared with pH 7.4 in strain PQ37. The influence of SOD (9.1 Marklund units per assay) is, however, different at the 2 p H conditions of the test.
500
400 ¢o
300
~2oo i lOO
L 16.5
3~3
Quercetin,
, 49.5
66
nmol
Fig. 1. Dose-response curves in the Ames assay (strain TA98) of quercetin (open squares) and quercetin in the presence of 13 Marklund units SOD (closed squares), $9 without cofactors (open circles), $100 without cofactors (open triangles), $9 with cofactors (closed circles) and S100 with cofactors (closed triangles). Each point is the average of at least 2 independent experiments.
~.
3
g
2
'6
c t/) O o~
pH 7.4
pH 7.4 pH 6.0 pH 6.0 + SOD + SOD Fig. 2. SOS-induction factors+SD (average of at least 2 independent experiments) of 16.5 nmole quercetin in the SOS chromotest (strain PQ37) at pH 7.4 and 6.0 in the absence or the presence of SOD (9.1 Marklund units per assay). The SOS induction factor l(c) at concentration c is: l(c)= R(c)/R(O), where R is the ratio /3-galactosidase activity/alkaline phosphatase activity.
At p H 6.0 the presence of SOD, which is stable in the p H range tested (Fridovich, 1989), does not result in an enhancement of SOS response, whereas it does at p H 7.4. One possible mechanism to explain the enhancing effect of SOD on the induction of SOS functions by quercetin at p H 7.4 could be the subsequent production of H 2 0 2 as a result of dismutation of superoxide which is spontaneously produced at p H 7.4 by autooxidation of quercetin (Rueff et al., 1989), although Brawn and Fridovich (1985) have shown that superoxide radical can be a possible inducer of SOS functions in E. coli. Hydrogen peroxide is an inducer of SOS functions in strain PQ37 (Fig. 3), seemingly through production of DNA-strand breaks (Goerlich et al., 1989). This response in the SOS chromotest was due to a net increase in/3-galactosidase activity (data not shown). Induction of SOS functions by H 2 0 2 occurs at concentrations approximately 4 times higher than quercetin. Moreover, in the assays of quercetin carried out in the presence of SOD and catalase
79
15 13
11
9 4--
7
.E
5
0 3 1
k 0
0.2
I
J
0.4
0.6
I
H202,
0.8
mM
Fig. 3. D o s e - r e s p o n s e curve of H2O 2 in the SOS chromotest (strain PQ37). Each point is the average of at least 2 independent experiments. The SOS-inducing activity was the result of a net increase in/3-galactosidase activity.
no differences in the SOS-inducing activity were found. H 2 0 ~ might thus not be the ultimate SOS inducer resulting from the effect of SOD on quercetin. Discussion
The results reported in the present work were carried out to help ascertain the different effects of $9 on the activity of quercetin in the Ames assay versus other short-term assays. Quercetin is degraded by self-produced superoxide anion at pH values above neutrality (Ueno et al., 1984; Rueff et al., 1989). It is thus conceivable to envisage the enhancing effect of $9 on the mutagenicity of quercetin in the Ames assay as the result of protection afforded by the SOD present in $9. As shown in Table 1, $9 contains 7.1 SOD u n i t s / m g protein. Yet, from our data on the effect of SOD on the mutagenicity of quercetin (Table 2 and Fig. 1) it can be unambiguously concluded that SOD does not justify per se the enhancing effect of $9. Neither does it do so for the cytosolic fraction (S100) which con-
tains 22 SOD units/mg protein (Table 1) as can be concluded from the data presented in Fig. 1. SOD has also been shown to enhance the comutagenicity of norharman (Nagao et al., 1986). As regards quercetin, the data from these authors (Ochiai et al., 1984; Nagao et al., 1986) clearly indicate that besides SOD there are other factors in the cytosolic fraction which contribute to its enhancing effect on the mutagenicity of quercetin. Vrijsen et al. (1990) suggest that the effect of SI00 might be entirely due to protection of quercetin against oxidative degradation. It might reasonably be so in view of our results (Fig. 1). As a matter of fact, S100 whether in the presence or in the absence of cofactors exerts a consistent enhancing effect on the mutagenicity of quercetin, and such an effect is definitely not attributable to any cytochrome P450-dependent metabolism. This is based on the undetectable levels of cytochrome P450 in S100 (Table 1) and on the absence of any role of cofactors in the enhancing effect of S100 (Fig. 1). Cytochrome P450-dependent metabolization of quercetin seems to account for part of the enhancing effect of $9 (Fig. 1) since the dose-response curves with and without cofactors are significantly different ( p < 0.05). It should be noted when comparing the mutagenicity of quercetin with $9 and S100 that the activities of cytosolic enzymes are concentrated in the latter (Table 1). This holds true for SOD and catalase and could apply to other cytosolic factors (Ochiai et al., 1984) which enhance the mutagenicity of quercetin. The SOS-inducing activity of quercetin is pHdependent with a higher activity at acidic pH (Fig. 2). Hatcher and Bryan (1985) have reported on a similar dependence in the Ames test. According to those authors this effect could be due to a decreased ionization of quercetin at lower pH values, thereby increasing its uptake by the tester strains, or to the stability of the molecule at acidic pH values, or to a combination of these 2 mechanisms. Since SOD has no effect on the SOS-inducing activity of quercetin at pH 6.0, but enhances its activity at pH 7.4 (Fig. 2), it would seem likely that quercetin itself is the ultimate inducer of SOS functions at pH levels where no autooxidation occurs.
80
The convertogenic activity of plant phenolics in Saccharornyces cerevisiae could only be detected at alkaline pH values. This phenomenon was interpreted as the result of the autooxidation of phenolics under alkaline conditions, which leads to the generation of H 202 and free radicals (Rosin, 1984). In CHO cells it has been noted that the pattern of genetic damage by quercetin is similar to that of ionizing radiation suggesting the possibility of free radical-induced lesions (Carver et al., 1983; MacGregor, 1986). The superoxide anion produced by the autooxidation of quercetin cannot, as such, freely cross biological membranes and thus contribute to a measurable genotoxic effect. The protonated form of superoxide (HO:~), however, is able to cross membranes as effectively as H20 2. Only a minor amount of superoxide exists in the form of HO 2 at neutral pH values, but that amount might be fairly high in the proximity of membranes. The superoxide anion could thus only indirectly display genotoxic activity. Dismutation of superoxide gives rise to H20 2 which can damage DNA. Usage of scavenging enzymes (e.g., SOD, catalase) would thus result in lower responses for quercetin when the genetic end-points for which the activity is assessed respond preferentially to the oxygen species derived from quercetin (Rueff et al., 1989). This might be the, case for chromosomal aberrations, sister-chromatid exchanges and mutations at the tk locus (Meltz and MacGregor, 1981; Carver et al., 1983), although there is still no direct proof for this mechanism. Since, however, quercetin is degraded in the autooxidative process, the effect of scavenging enzymes results in higher responses of quercetin for genetic end-points for which the flavonol by itself is the genotoxicant. This could be the case for the induction of SOS functions at pH 7.4 and also partially explain the activity in the Ames assay, although in the latter the SOD present in $9 clearly does not account entirely for the enhancement, suggesting either metabolic activation by $9 or the presence of additional antioxidant defenses in $9. The mechanism(s) of DNA damage by quercetin remains by and large to be elucidated, and an understanding of those mechanisms could help in interpreting the controversial results in
carcinogenicity testing of the flavonol (Pamucku et al., 1980; Hirono et al., 1981). The various patterns of genotoxicity of quercetin for different genetic end-points, and possibly its unproven carcinogenicity are, on the basis of the available evidence, subject to a wealth of factors (pH, antioxidants, metabolic pathways) whose precise role in the particular conditions of each test would deserve further study.
Acknowledgements We are grateful to Prof. J. Mexia. for his invaluable assistance with the statistical analysis. Our current research is supported by the LusoAmerican Foundation for Development, the Commission of the European Communities (EV4V 0069) and the Calouste Gulbenkian Foundation. J.G. and A.R. are supported by a doctoral fellowship from JNICT.
References Bjeldanes, L.F., and G.W. Chang (1977) Mutagenic activity of quercetin and related compounds, Science, 197, 577-578. Brawn, M.K., and I. Fridovich (1985) Increased superoxide radical production evokes inducible DNA repair in Escherichia coli, J. Biol. Chem., 260, 922-925. 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. Carver, J.H., A.V. Carrano and J.T. MacGregor (1983) Genetic effects of flavonols quercetin, kaempferol and galangin on Chinese hamster ovary cells in vitro, Mutation Res., 113, 45-60. De Flora, S., C. Bennicelli, P. Zanacchi, F.D. Agostini and A. Camoirano (1989) Mutagenicity of active oxygen species in bacteria and its enzymatic or chemical inhibition, Mutation Res., 214, 153-158. Fridovich, I. (1989) Superoxide dismutases - an adaptation to a paramagnetic gas, J. Biol. Chem., 264, 7761-7764. Goerlich, O., P. Quillardet and M. Hofnung (1989) Induction of SOS response by peroxide in various Escherichia coli mutants with altered protection against oxidative DNA damage, J. Bacteriol., 171, 6141-6147. Hatcher, J.F., and G.T. Bryan (1985) Factors affecting the mutagenic activity of quercetin for Salmonella typhimurium TA98: metals ions, antioxidants and pH, Mutation Res., 148, 13-23. Hirono, I., I. Ueno, S. Hosaka, H. Takanashi, T. Matsushima, T. Sugimura and S. Natori (1981) Carcinogenicity examination of quercetin and rutin in ACI rats, Cancer Lett., 13, 15-21.
81 Laires, A., H. Borba, J. Rueff, I. Gomes and M. Halpern (1982) Urinary mutagenicity in occupational exposure to mineral oils and iron oxide particles, Carcinogenesis, 3, 1077-1079. Llagostera, M., S. Garrido, J. Barbe, R. Guerrero and J. Rueff (1987) Influence of $9 mix in the induction of SOS system by quercetin, Mutation Res., 191, 1-4. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall (1951) Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193, 265-275. MacGregor, J.T. (1986) Genetic toxicology of dietary flavonoids, in: I. Knudsen (Ed.), Genetic Toxicology of the Diet, Liss, New York, pp. 33-43. MacGregor, J.T., and L. Jurd (1978) Mutagenicity of plant flavonoids: structural requirements for mutagenic activity in Salmonella typhimurium, Mutation Res., 54, 297-309. Marklund, S., and G. Marklund (1974) Involvement of the superoxide anion radical in the autooxidation 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. Marzin, D.R., P. Olivier and H. Vophi (1986) Kinetic determination of enzymatic activity and modification of metabolic activation system in the SOS chromotest, Mutation Res., 164, 353-359. Meltz, M.L., and J.T. MacGregor (1981) Activity of the plant flavonol quercetin in the mouse lymphoma L5178Y TK ÷/ mutation, DNA single strand break, and BALB/c3T3 chemical transformation assay, Mutation Res., 88, 317-324. Nagao, M., K. Wakabayashi, Y. Suwa and T. Kobayashi (1986) Alteration of mutagenic potentials by peroxidase, catalase and superoxide dismutase, in: D.M. Shankel, P.E. Hartman, T. Kada and A. Hollaender (Eds), Antimutagenesis and Anticarcinogenesis Mechanisms, Plenum, New York, pp. 73-80.
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. Omura, T., and R. Sato (1964) The carbon monoxide-binding pigment of liver microsomes. 11. Solubilization, purification, and properties, J. Biol. Chem., 239, 2379-2385. Pamucku, A.M., S. Yalciner, J.F. Hatcher and G.T. Bryan (1980) Quercetin, a rat intestinal and bladder carcinogen present in bracken fern (Pteridium aquilinum), Cancer Res., 40, 3468-3472. Quillardet, P., and M. Hofnung (1985) The SOS Chromotest, a colorimetic bacterial assay for genotoxins: procedures, Mutation Res., 147, 65-78. Rosin, P.R. (1984) The influence of pH on the convertogenic activity of plant phenolics, Mutation Res., t35, 109-113. Rueff, J., A. Laires, H. Borba, T. Chaveca, M.I. Gomes and M. Halpern (1986) Genetic toxicology of flavonoids: the role of metabolic conditions in the induction of reverse mutation, SOS functions and sister-chromatid exchanges, Mutagenesis, 1, 179-183. Rueff, J., A. Laires, A. Br~s, H. Borba, T. Chaveca, J. Gaspar, A. Rodrigues, L. Cristov~o and M. Monteiro (1989) DNA damage and oxygen species, in: M.W. Lambert and J. Laval (Eds.), DNA Repair and their Biological Implications in Mammalian Cells, Plenum, New York, pp. 171181. Sinha, A.K. (1972) Colorimetric assay of catalase, Anal. Biochem., 47, 389-394. Ueno, I., M. Kohno, K. Haraikawa and I. Hirono (1984) Interaction between quercetin and superoxide radicals. Reduction of the quercetin mutagenicity, J. Pharm. Dyn., 7, 798-803. Vrijsen, R., Y. Michote and A. Boey6 (1990) Metabolic activation of quercetin mutagenicity, Mutation Res., 232, 243248.
75
© 1992 Elsevier Science Publishers B.V. All rights reserved 0027-5107/92/$05.00
MUT 05035
Oxygen species and the genotoxicity of quercetin Jose Rueff 1, Ant6nio Laires 1,2,Jorge Gaspar l, Helena Borba 1 and Ant6nio Rodrigues 1 1 Department of Genetics, Faculty of Medical Sciences UNL, Lisbon and 2 Faculty of Sciences and Technology UNL, Monte da Caparica (Portugal)
(Received 3 April 1991) (Revision received 10 July 1991) (Accepted 22 July 1991)
Keywords: Quercetin; Oxygen species; SOS induction
Summary Quercetin has been extensively studied in various short-term assays for genotoxicity. The patterns of genotoxicity of quercetin for different genetic endpoints are subject to a variety of factors (pH, antioxidants, metabolism) whose precise role in each test remains unclear. In the present study we report on the possible effect of oxygen-derived species on the activity of quercetin in the Ames assay and in the SOS chromotest. Our results seem to suggest that superoxide dismutase (SOD) does not account for the levels of mutagenicity detected in the presence of $9 or S100. The latter may, however, contain other factors of antioxidant defense which may prevent the oxidative degradation of quercetin. Since this degradation occurs at pH values above neutrality and the SOS-inducing activity is higher at pH 6.0, it is concluded that the response of quercetin in the SOS chromotest is due to quercetin itself at acidic pH. The SOS-inducing activity at pH 7.4 is enhanced by SOD, but it cannot be unambiguously concluded that this effect in the SOS chromotest might only be due to protection against the oxidative degradation of quercetin.
D N A damage by quercetin may occur via more than one mechanism. Reports have appeared on the influence of the mammalian microsome system ($9) on the activity of quercetin towards various genetic end-points. The results obtained using the Ames assay, the induction of sisterchromatid exchanges in human lymphocytes and
Correspondence: Dr. J. Rueff, Department of Genetics, Faculty of Medical Sciences UNL, R. Junqueira 96, P-1300 Lisbon (Portugal).
the induction of some of the genes of the SOS system in E. coli (suM, recA and u m u C ) seem to point to the conclusion that quercetin exerts its genotoxic activity either directly or through putative degradation products depending on the genetic end-point assessed (Rueff et al., 1986, 1989; Llagostera et al., 1987). In the L5178Y TK +/- system (Meltz and MacGregor, 1981) the mutagenicity of quercetin has been shown to be lowered by the presence of $9, and in C H O cells quercetin was likewise genotoxic only in the' absence of $9 (Carver et al., 1983).
76
In the Ames assay, however, the mutagenicity of quercetin is consistently enhanced by the presence of $9 (Bjeldanes and Chang, 1977; MacGregor and Jurd, 1978; Brown and Dietrich, 1979; Hatcher and Bryan, 1985; Rueff et al., 1986; Vrijsen et al., 1990). The enhancement of the mutagenicity of quercetin in the Ames assay has been shown to depend on the cytosolic (S100) fraction present in $9 rather than on microsomal enzymes (Brown and Dietrich, 1979; Ochiai et al., 1984; Rueff et al., 1986). The possible metabolite(s) of quercetin which might be responsible for the enhancement of mutagenicity in the Ames assay still await identification. Yet, quercetin spontaneously gives rise to the superoxide anion as a function of increasing pH values and is degraded in this autooxidative process with formation of a product detectable by HPLC (Ueno et al., 1984; Rueff et al., 1989). These observations may lead to 2 preliminary hypotheses: (i) in the Ames assay the mutagenicity of quercetin is enhanced due to protection afforded by the copper-zinc superoxide dismutase (SOD) present in $9 (Ochiai et al., 1984); (ii) in other assay systems, e.g., SOS chromotest, sister-chromatid exchanges, chromosomal aberrations, the genotoxicity might be due instead to oxygen-derived species produced by spontaneous degradation of quercetin, which would explain the decreased response in the presence of $9 (Carver et al., 1983; Rueff et al., 1986). Vrijsen et al. (1990) have recently reported that $9, but not S100, is capable of metabolizing quercetin which could help in explaining the enhancement of activity in the Ames assay. Along this line we report here on additional studies performed to address the hypotheses put forward above.
contents in the $9 and S100 fractions were determined according to Lowry et al. (1951). Enzyme assays Superoxide dismutase (SOD) activities in $9 and S100 were determined as described by Marklund and Marklund (1974). One enzyme unit is the amount of protein that inhibits the autooxidation of pyrogallol at pH 8.0 by 50%, followed at 420 nm. Catalase (CAT) activities in $9 and S100 were determined as described by Sinha (1972). The method is based on the reduction of potassium dichromate in acetic acid to chromic acetate when heated in the presence of hydrogen peroxide, measured at 570 nm. The catalase contents of $9 and S100 were expressed in terms of rate constant (Kat) per g of protein. Cytochrome P450 contents of $9 and S100 were determined from CO difference spectra of dithionite-reduced samples of $9 and S100 according to Omura and Sato (1964).
Materials and methods
Ames test Salmonella typhimurium TA98, obtained from Professor B.N. Ames (Berkeley, CA), was used as tester strain. Mutagenicity testing in the Ames assay was carried out as previously described (Maron and Ames, 1983; Rueff et al., 1986). For the assays carried out in the presence of SOD (EC 1.1.5.1.1) and catalase (EC 1.11.1.6), both from Sigma, the enzymes were prepared, respectively, in phosphate buffer 0.1 M, pH 7.4 and 0.02 M, pH 7.4. Diagnostic mutagens were 2-nitrofluorene (4.7 nmole/plate without $9) and benzo[a]pyrene (19.8 nmole/plate). At least 2 independent experiments were carried out for each dose level. Mutagenic activity was determined from the slope of the least squares line of the linear portion of the dose-response curve.
Rat liver enzymes $9 was prepared as described previously (Laires et al., 1982; Maron and Ames, 1983). The S100 fraction was prepared by centrifuging fresh $9 for 1 h at 100,000 x g. The supernatants were used routinely in $9 mix and S100 mix, without and with cofactors (glucose-6-phosphate and NADP) at a level of 0.1 ml per 1.0 ml of mix. The protein
SOS chromotest Induction of SOS functions was assayed with the SOS chromotest by determining the induced levels of fl-galactosidase whose gene, lacZ, had been placed under the control of sulA, one of the SOS genes involved in cell division inhibition (Quillardet and Hofnung, 1985). The strain Escherichia coli PQ37 was obtained from Professor
77
M. Hofnung (Paris). The SOS chomotest was performed as described by Quillardet and Hofnung (1985) with the adaptation introduced by Marzin et al. (1986). This consisted of kinetic measurements of /3-galactosidase and alkaline phosphatase activities instead of 'end-point' assays. When testing the SOS-inducing activity of quercetin at pH 6.0 or pH 7.4 the dilution 1 : 10 of the culture was made in LB/phosphate buffer, pH 6.0 or pH 7.4, 1:1, respectively. For the assays carried out in the presence of SOD (9.1 Marklund units per assay) the enzyme was prepared in 0.2 M phosphate buffers, pH 6.0 and pH 7.4. For the assays carried out in the presence of catalase (14 units per assay), the enzyme was prepared in 0.02 M phosphate buffer, pH 7.4. The diagnostic mutagen was 4-nitroquinoline-Noxide (1.05 nmole/assay). At least 2 independent experiments were carried out for each dose level. Results
The catalase and superoxide dismutase activities in $9 and S100 used in the present study are presented in Table 1 along with the cytochrome P450 content. The experiments carried out to ascertain the role of catalase and superoxide dismutase on the mutagenic activity of 33 nmole quercetin in strain TA98 are presented in Table 2 along with the response in the presence of $9 and S100. Catalase has no effect on the mutagenicity of
TABLE 1 E N Z Y M A T I C ACTIVITIES O F S U P E R O X 1 D E DISMUT A S E (SOD), C A T A L A S E (CAT) A N D Cyt P450 1N $9 A N D S100 $9 SOD ( u n i t s / m g protein) CAT ( K a t / g protein) Cyt P450 ( n m o l e / m g protein)
S100 7.1
22.0
292
4211
0.77
n.d.
n.d., not detectable.
quercetin. SOD at a level higher than present in S100 or $9 (30 Marklund units vs. 22 in S100 and 13 in $9) exerts an enhancing effect on the mutagenicity of quercetin albeit lower than that observable with $9 or S100. The superoxide anion does not respond in strain TA98 as shown in Table 2 using the xanthine/xanthine oxidase system. The absence of response of superoxide is in agreement with the results of Ueno et al. (1984) and De Flora et al. (1989). The xanthine/ xanthine oxidase system completely abolishes the mutagenicity of quercetin through degradation by superoxide (Table 2). Dose-response curves of quercetin in the presence of 13 units SOD, and in the presence of $9 mix, S100 mix and these 2 extracts with added cofactors are presented in Fig. 1.
TABLE 2 R O L E O F S U P E R O X 1 D E D I S M U T A S E (SOD), C A T A L A S E (CAT) A N D X A N T H 1 N E / X A N T H I N E O X I D A S E ( X / X O ) ON T H E M U T A G E N I C I T Y O F Q U E R C E T I N ~' Control Quercetin (0 nmole) Quercetin (33 nmole)
$9 mix b (500 ~1)
S100 mix c (500 p.I)
SOD (30 U) 0
CAT (100 U) ~
X/XO r
29
46
27
25
31
35
120
276
247
164
127
30
R e v . / p l a t e in strain TA98. Three independent experiments. b 13 S O D Marklund u n i t s / 5 0 0 ~ l $9 mix. c 22 S O D Marklund u n i t s / 5 0 0 ~ l $100 mix. d Marklund units. O n e unit decomposes 1 /~mole of H 2 0 2 / m i n at pH 7.0 and 2 5 ° C . f X = 15 ~ g / p l a t e ; XO = 0.0021 u n i t s / p l a t e .
78
Statistical analysis of the regression output (F-test, multiple comparison of Scheff6) shows that quercetin mutagenicity is not significantly increased in the presence of SOD. Thus, SOD at levels higher than present in $9 does not afford enough protection against the autooxidative degradation of quercetin. Both $9 mix and S100 mix significantly enhanced quercetin mutagenicity ( p < 0.05). When $9 mix is prepared without the cofactors needed for cytochrome P450-mediated metabolism the response is significantly lower ( p < 0.05). The absence of cofactors in $100 mix, however, does not result in any statistically significant effect as compared with S100 with cofactors. Both $9 mix and S100 mix in the absence of cofactors exert the same enhancing effect on the mutagenicity of quercetin. As shown in Fig. 2 the SOS-inducing activity of quercetin is significantly higher at p H 6.0 as compared with pH 7.4 in strain PQ37. The influence of SOD (9.1 Marklund units per assay) is, however, different at the 2 p H conditions of the test.
500
400 ¢o
300
~2oo i lOO
L 16.5
3~3
Quercetin,
, 49.5
66
nmol
Fig. 1. Dose-response curves in the Ames assay (strain TA98) of quercetin (open squares) and quercetin in the presence of 13 Marklund units SOD (closed squares), $9 without cofactors (open circles), $100 without cofactors (open triangles), $9 with cofactors (closed circles) and S100 with cofactors (closed triangles). Each point is the average of at least 2 independent experiments.
~.
3
g
2
'6
c t/) O o~
pH 7.4
pH 7.4 pH 6.0 pH 6.0 + SOD + SOD Fig. 2. SOS-induction factors+SD (average of at least 2 independent experiments) of 16.5 nmole quercetin in the SOS chromotest (strain PQ37) at pH 7.4 and 6.0 in the absence or the presence of SOD (9.1 Marklund units per assay). The SOS induction factor l(c) at concentration c is: l(c)= R(c)/R(O), where R is the ratio /3-galactosidase activity/alkaline phosphatase activity.
At p H 6.0 the presence of SOD, which is stable in the p H range tested (Fridovich, 1989), does not result in an enhancement of SOS response, whereas it does at p H 7.4. One possible mechanism to explain the enhancing effect of SOD on the induction of SOS functions by quercetin at p H 7.4 could be the subsequent production of H 2 0 2 as a result of dismutation of superoxide which is spontaneously produced at p H 7.4 by autooxidation of quercetin (Rueff et al., 1989), although Brawn and Fridovich (1985) have shown that superoxide radical can be a possible inducer of SOS functions in E. coli. Hydrogen peroxide is an inducer of SOS functions in strain PQ37 (Fig. 3), seemingly through production of DNA-strand breaks (Goerlich et al., 1989). This response in the SOS chromotest was due to a net increase in/3-galactosidase activity (data not shown). Induction of SOS functions by H 2 0 2 occurs at concentrations approximately 4 times higher than quercetin. Moreover, in the assays of quercetin carried out in the presence of SOD and catalase
79
15 13
11
9 4--
7
.E
5
0 3 1
k 0
0.2
I
J
0.4
0.6
I
H202,
0.8
mM
Fig. 3. D o s e - r e s p o n s e curve of H2O 2 in the SOS chromotest (strain PQ37). Each point is the average of at least 2 independent experiments. The SOS-inducing activity was the result of a net increase in/3-galactosidase activity.
no differences in the SOS-inducing activity were found. H 2 0 ~ might thus not be the ultimate SOS inducer resulting from the effect of SOD on quercetin. Discussion
The results reported in the present work were carried out to help ascertain the different effects of $9 on the activity of quercetin in the Ames assay versus other short-term assays. Quercetin is degraded by self-produced superoxide anion at pH values above neutrality (Ueno et al., 1984; Rueff et al., 1989). It is thus conceivable to envisage the enhancing effect of $9 on the mutagenicity of quercetin in the Ames assay as the result of protection afforded by the SOD present in $9. As shown in Table 1, $9 contains 7.1 SOD u n i t s / m g protein. Yet, from our data on the effect of SOD on the mutagenicity of quercetin (Table 2 and Fig. 1) it can be unambiguously concluded that SOD does not justify per se the enhancing effect of $9. Neither does it do so for the cytosolic fraction (S100) which con-
tains 22 SOD units/mg protein (Table 1) as can be concluded from the data presented in Fig. 1. SOD has also been shown to enhance the comutagenicity of norharman (Nagao et al., 1986). As regards quercetin, the data from these authors (Ochiai et al., 1984; Nagao et al., 1986) clearly indicate that besides SOD there are other factors in the cytosolic fraction which contribute to its enhancing effect on the mutagenicity of quercetin. Vrijsen et al. (1990) suggest that the effect of SI00 might be entirely due to protection of quercetin against oxidative degradation. It might reasonably be so in view of our results (Fig. 1). As a matter of fact, S100 whether in the presence or in the absence of cofactors exerts a consistent enhancing effect on the mutagenicity of quercetin, and such an effect is definitely not attributable to any cytochrome P450-dependent metabolism. This is based on the undetectable levels of cytochrome P450 in S100 (Table 1) and on the absence of any role of cofactors in the enhancing effect of S100 (Fig. 1). Cytochrome P450-dependent metabolization of quercetin seems to account for part of the enhancing effect of $9 (Fig. 1) since the dose-response curves with and without cofactors are significantly different ( p < 0.05). It should be noted when comparing the mutagenicity of quercetin with $9 and S100 that the activities of cytosolic enzymes are concentrated in the latter (Table 1). This holds true for SOD and catalase and could apply to other cytosolic factors (Ochiai et al., 1984) which enhance the mutagenicity of quercetin. The SOS-inducing activity of quercetin is pHdependent with a higher activity at acidic pH (Fig. 2). Hatcher and Bryan (1985) have reported on a similar dependence in the Ames test. According to those authors this effect could be due to a decreased ionization of quercetin at lower pH values, thereby increasing its uptake by the tester strains, or to the stability of the molecule at acidic pH values, or to a combination of these 2 mechanisms. Since SOD has no effect on the SOS-inducing activity of quercetin at pH 6.0, but enhances its activity at pH 7.4 (Fig. 2), it would seem likely that quercetin itself is the ultimate inducer of SOS functions at pH levels where no autooxidation occurs.
80
The convertogenic activity of plant phenolics in Saccharornyces cerevisiae could only be detected at alkaline pH values. This phenomenon was interpreted as the result of the autooxidation of phenolics under alkaline conditions, which leads to the generation of H 202 and free radicals (Rosin, 1984). In CHO cells it has been noted that the pattern of genetic damage by quercetin is similar to that of ionizing radiation suggesting the possibility of free radical-induced lesions (Carver et al., 1983; MacGregor, 1986). The superoxide anion produced by the autooxidation of quercetin cannot, as such, freely cross biological membranes and thus contribute to a measurable genotoxic effect. The protonated form of superoxide (HO:~), however, is able to cross membranes as effectively as H20 2. Only a minor amount of superoxide exists in the form of HO 2 at neutral pH values, but that amount might be fairly high in the proximity of membranes. The superoxide anion could thus only indirectly display genotoxic activity. Dismutation of superoxide gives rise to H20 2 which can damage DNA. Usage of scavenging enzymes (e.g., SOD, catalase) would thus result in lower responses for quercetin when the genetic end-points for which the activity is assessed respond preferentially to the oxygen species derived from quercetin (Rueff et al., 1989). This might be the, case for chromosomal aberrations, sister-chromatid exchanges and mutations at the tk locus (Meltz and MacGregor, 1981; Carver et al., 1983), although there is still no direct proof for this mechanism. Since, however, quercetin is degraded in the autooxidative process, the effect of scavenging enzymes results in higher responses of quercetin for genetic end-points for which the flavonol by itself is the genotoxicant. This could be the case for the induction of SOS functions at pH 7.4 and also partially explain the activity in the Ames assay, although in the latter the SOD present in $9 clearly does not account entirely for the enhancement, suggesting either metabolic activation by $9 or the presence of additional antioxidant defenses in $9. The mechanism(s) of DNA damage by quercetin remains by and large to be elucidated, and an understanding of those mechanisms could help in interpreting the controversial results in
carcinogenicity testing of the flavonol (Pamucku et al., 1980; Hirono et al., 1981). The various patterns of genotoxicity of quercetin for different genetic end-points, and possibly its unproven carcinogenicity are, on the basis of the available evidence, subject to a wealth of factors (pH, antioxidants, metabolic pathways) whose precise role in the particular conditions of each test would deserve further study.
Acknowledgements We are grateful to Prof. J. Mexia. for his invaluable assistance with the statistical analysis. Our current research is supported by the LusoAmerican Foundation for Development, the Commission of the European Communities (EV4V 0069) and the Calouste Gulbenkian Foundation. J.G. and A.R. are supported by a doctoral fellowship from JNICT.
References Bjeldanes, L.F., and G.W. Chang (1977) Mutagenic activity of quercetin and related compounds, Science, 197, 577-578. Brawn, M.K., and I. Fridovich (1985) Increased superoxide radical production evokes inducible DNA repair in Escherichia coli, J. Biol. Chem., 260, 922-925. 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. Carver, J.H., A.V. Carrano and J.T. MacGregor (1983) Genetic effects of flavonols quercetin, kaempferol and galangin on Chinese hamster ovary cells in vitro, Mutation Res., 113, 45-60. De Flora, S., C. Bennicelli, P. Zanacchi, F.D. Agostini and A. Camoirano (1989) Mutagenicity of active oxygen species in bacteria and its enzymatic or chemical inhibition, Mutation Res., 214, 153-158. Fridovich, I. (1989) Superoxide dismutases - an adaptation to a paramagnetic gas, J. Biol. Chem., 264, 7761-7764. Goerlich, O., P. Quillardet and M. Hofnung (1989) Induction of SOS response by peroxide in various Escherichia coli mutants with altered protection against oxidative DNA damage, J. Bacteriol., 171, 6141-6147. Hatcher, J.F., and G.T. Bryan (1985) Factors affecting the mutagenic activity of quercetin for Salmonella typhimurium TA98: metals ions, antioxidants and pH, Mutation Res., 148, 13-23. Hirono, I., I. Ueno, S. Hosaka, H. Takanashi, T. Matsushima, T. Sugimura and S. Natori (1981) Carcinogenicity examination of quercetin and rutin in ACI rats, Cancer Lett., 13, 15-21.
81 Laires, A., H. Borba, J. Rueff, I. Gomes and M. Halpern (1982) Urinary mutagenicity in occupational exposure to mineral oils and iron oxide particles, Carcinogenesis, 3, 1077-1079. Llagostera, M., S. Garrido, J. Barbe, R. Guerrero and J. Rueff (1987) Influence of $9 mix in the induction of SOS system by quercetin, Mutation Res., 191, 1-4. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall (1951) Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193, 265-275. MacGregor, J.T. (1986) Genetic toxicology of dietary flavonoids, in: I. Knudsen (Ed.), Genetic Toxicology of the Diet, Liss, New York, pp. 33-43. MacGregor, J.T., and L. Jurd (1978) Mutagenicity of plant flavonoids: structural requirements for mutagenic activity in Salmonella typhimurium, Mutation Res., 54, 297-309. Marklund, S., and G. Marklund (1974) Involvement of the superoxide anion radical in the autooxidation 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. Marzin, D.R., P. Olivier and H. Vophi (1986) Kinetic determination of enzymatic activity and modification of metabolic activation system in the SOS chromotest, Mutation Res., 164, 353-359. Meltz, M.L., and J.T. MacGregor (1981) Activity of the plant flavonol quercetin in the mouse lymphoma L5178Y TK ÷/ mutation, DNA single strand break, and BALB/c3T3 chemical transformation assay, Mutation Res., 88, 317-324. Nagao, M., K. Wakabayashi, Y. Suwa and T. Kobayashi (1986) Alteration of mutagenic potentials by peroxidase, catalase and superoxide dismutase, in: D.M. Shankel, P.E. Hartman, T. Kada and A. Hollaender (Eds), Antimutagenesis and Anticarcinogenesis Mechanisms, Plenum, New York, pp. 73-80.
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. Omura, T., and R. Sato (1964) The carbon monoxide-binding pigment of liver microsomes. 11. Solubilization, purification, and properties, J. Biol. Chem., 239, 2379-2385. Pamucku, A.M., S. Yalciner, J.F. Hatcher and G.T. Bryan (1980) Quercetin, a rat intestinal and bladder carcinogen present in bracken fern (Pteridium aquilinum), Cancer Res., 40, 3468-3472. Quillardet, P., and M. Hofnung (1985) The SOS Chromotest, a colorimetic bacterial assay for genotoxins: procedures, Mutation Res., 147, 65-78. Rosin, P.R. (1984) The influence of pH on the convertogenic activity of plant phenolics, Mutation Res., t35, 109-113. Rueff, J., A. Laires, H. Borba, T. Chaveca, M.I. Gomes and M. Halpern (1986) Genetic toxicology of flavonoids: the role of metabolic conditions in the induction of reverse mutation, SOS functions and sister-chromatid exchanges, Mutagenesis, 1, 179-183. Rueff, J., A. Laires, A. Br~s, H. Borba, T. Chaveca, J. Gaspar, A. Rodrigues, L. Cristov~o and M. Monteiro (1989) DNA damage and oxygen species, in: M.W. Lambert and J. Laval (Eds.), DNA Repair and their Biological Implications in Mammalian Cells, Plenum, New York, pp. 171181. Sinha, A.K. (1972) Colorimetric assay of catalase, Anal. Biochem., 47, 389-394. Ueno, I., M. Kohno, K. Haraikawa and I. Hirono (1984) Interaction between quercetin and superoxide radicals. Reduction of the quercetin mutagenicity, J. Pharm. Dyn., 7, 798-803. Vrijsen, R., Y. Michote and A. Boey6 (1990) Metabolic activation of quercetin mutagenicity, Mutation Res., 232, 243248.
