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Teratogenesis, Carcinogenesis, and Mutagenesis 16:229-241(1996)
Mutagenicity of Kaempferol in V79 Cells: The Role of Cytochromes P450’
1. Duarte Silva, A. Rodrigues, J. Gaspar, R. Maia, A. Laires, and J. Rueff Department of Genetics, Faculty of Medical Sciences (I. D.S., A. R., J.G., R. M., A. L., J. R.), ISMAG (A. R.), and Faculty of Sciences and Technology (A. L.), New University of tisbon
The flavonol kaempferol is widely found in the diet and i s directly mutagenic in some short-term tests, such as the induction of chromosomal aberrations in eukary- otic cells. The presence of exogenous metabolizing systems enhances its mutagenic- ity. We have evaluated the role of cytochromes P450 in the induction of chromosomal aberrations by kaempferol in V79 cells. The results obtained suggest that there is a time- dependent biotransformation of kaempferol to quercetin, by cytochromes P4.50, as as- sessed by high pressure liquid chromatography. Quercetin seems to contribute to the mutagenicity of kaempferol in the presence of microsomal metabolizing systems. On the other hand, the direct induction of chromosomal aberrations by kaempferol does not seem to depend on the production of reactive oxygen species. o 1997 Wiiey-Liss, Inc.
Key words: kaempferol, V79 cells, chromosomal aberrations, cytochromes P450, mutagenicity, quercetin
INTRODUCTION
The epidemiological studies of Doll and Pet0 [ 11 have suggested that diet is one of the most important causes of cancer deaths in the United States. Diet contains various mutagens and carcinogens that can be classified into three groups: naturally occurring chemicals, synthetic added contaminant compounds, and compounds pro- duced during the cooking process [2]. Toxicological evaluation of synthetic chemi- cals without a similar evaluation of naturally occurring chemicals has generated an imbalance in both data and perception about potential “natural hazards” to humans [ 3 ] . In this perspective the toxicological evaluation of natural cheinicals present in foodstuffs for their potential mutagenicity and carcinogenicity is important in order to have the right perception of the importance of natural vs. synthetic mutagens in food.
Address reprint requests to JosC Rueff, Department of Genetics, Faculty of Medical Sciences, New University of Lisbon, Rua da Junqueira 96, P-1300, Lisbon, Portugal.
‘Dedicated to Professor Luis Archer on the occasion of his 70th birthday.
0 1997 Wiley-Liss, Inc.
230 Duarte Silva et al.
Among natural products flavonoids represent one of the most numerous groups of natural compounds and are found in the edible portions of the majority of food plants [4]. Most of the plant flavonoids are in the form of glycosides. For quercetin, the most common flavonoid, about 70 glycosidic combinations have been character- ized and analyzed. Nearly as many glycosides have been described for the two other most common flavonols, kaempferol and myricetin [4].
Quercetin is the only flavonoid that has been tested for carcinogenicity and the results obtained are equivocal with some results being positive [5-71 whereas others are negative [8].
There are numerous studies on the mutagenicity of quercetin in the prokaryotic and eukaryotic cells [9-181. Only a few studies, however, report on the mutagenicity of kaempferol. Kaempferol, in the presence of a rat liver metabolizing system (S9 Mix), has been shown to induce mutations in Salmonella typhimurium [ 10,14] and in Drosophztu, as measured by the recessive sex-linked lethal test [ 151, in the tk locus of Chinese hamster ovary (CHO) cells, and to induce chromosomal aberrations in CHO cells both with and without metabolic activation [16]. Maruta et al. [17] have shown that kaempferol induces mutations at the hprt locus in mammalian V79 cells in the presence of S9 Mix. The identity of the metabolite is unknown.
In spite of the various studies available, the mechanisms of mutagenicity of kaempferol and quercetin are not fully understood. As polyphenols these products can, under some conditions, in particular alkaline pH values, produce reactive oxy- gen species (ROS) by autooxidation and therefore the involvement of ROS in their mutagenicity should be taken into account [13,18,19].
The present study was undertaken to further investigate the mechanisms of the mutagenicity of the flavonoid kaempferol in eukaryotic cells. V79 Chinese hamster cells were used as the test system and the induction of chromosomal aberrations (CAs) and micronuclei (MN) as the endpoints. We tested the influence of external metabolizing systems in the induction of CAs in order to evaluate the possible role of cytochromes P450 in its metabolism to more potent mutagenic products. We at- tempted to identify the metabolite(s) of the biotransformation of kaempferol by high pressure liquid chromatography (HPLC). The induction of CAs was also studied at different pH values and in the presence of the enzymes superoxide dismutase and catalase to evaluate the importance of ROS in its mutagenicity.
MATERIALS AND METHODS Chemicals and Culture Media
Kaempferol, mitomycin C, cyclophosphamide, superoxide dismutase from hu- man erythrocytes (SOD), catalase, newborn calf serum, and Ham’s F-10 medium were obtained from Sigma (St. Louis, MO). Dimethylsulfoxide (DMSO) and Gi- emsa dye were from Merck (Darmstadt, Germany). Colchicine was from Fluka (Buchs, Swizerland). Penicillin, streptomycin, and amphotericin B were from Irvine Scien- tific (Santa Ana, CA). Acetonitrile and acetic acid were from Merck (HPLC grade).
V79 Chinese Hamster Cells
V79 Chinese hamster cells were kindly provided by Professor H.R. Glatt (Mainz and Potsdam). Cells were cultured in 5 ml Ham’s F-10 medium supplemented with
Mutagenicity of Kaempferol in V79 Cells 231
10% newborn calf serum, penicillin (50 IU/ml), streptomycin (50 pg/ml), and am- photericin B (25 pg/ml) and incubated at 37°C under an atmosphere of 5% C02.
Rat Liver Extracts
S9 was prepared as described in detail elsewhere [20]. Briefly, male Wistar rats were injected with Aroclor 1254 (500 mg/kg) in corn oil. Five days later the rats were sacrificed by cervical elongation. The animals were fed ad libitum and 12 hr before sacrifice the food was removed. Livers were excised, washed extensively in KC1 0.154 M, homogenized in a Potter-Elvehjem, and centrifuged at 9,000 g. The supernatant was collected and stored at -80°C.
Microsomes were prepared by centrifuging the S9 at 100,000 g. The superna- tant (S100) was collected and stored at -80°C. The microsome pellet was also stored at -80°C. S9 Mix was prepared according to Maron and h i e s [2 1 3. CA Assay
Twenty hour cultures (approximately 1 x lo6 cells) were washed with Ham’s F- 10 medium reconstituted in phosphate buffer 0.01 M pH 7.4 and incubated in 5 ml of this medium for 2 hr in the presence of kaempferol (dissolved in DMSO, of which the final concentration did not exceed 56 mM). Mitomycin C (Sigma) at a concentration of 3 pM was used as a positive control. When metabolic activation was required, 500 pl of S9 Mix, 10% (v/v), and 4.5 ml of medium were used. Cyclophosphamide at a con- centration of 21.5 pM was used as a positive control. After the treatment, cells were washed with culture medium and grown for another 15 hr. Colchicine was added at a final concentration of 6 pglml, and cells were grown for a further 3 hr. Cells were then harvested by trypsinization. After a 3 min hypotonic treatment with KCI 0.56% (wlv), at 37OC, cells were fixed with methanollacetic acid (3:l) and slides were prepared and stained with Giemsa (4% in phosphate buffer 0.01 M pH 6.8) for 10 min. Two independent experiments were carried out with each test com- pound and 100 metaphases were scored for each dose level treatment group in each experiment. Scoring of the different types of aberrations followed the crite- ria described by Rueff et al. [22].
pH-Dependent Induction of CAs
These experiments were performed as described in “CA Assay,” but Ham’s F- 10 medium reconstituted in phosphate buffer 0.01 M pH 4.0, 7.4, or 8.0 was used during the 2 hr treatment period with 104.4 pM kaempferol (in 15 pl DMSO); 15 pl DMSO was added to control flasks. The pH values of the media after the 2 h incuba- tion period were 6.1,7.0, and 7.4, respectively.
Effect of SOD, Catalase, S100, S9, and Microsomes With and Without Cofactors on the Induction of CAs
These experiments were performed as described in ‘*CA Assay,” using 104.4 pM kaempferol (in 15 p1 DMSO), but in the presence of either 1) 500 pl of SlOO Mix, 2) S9 Mix without cofactors (NADP and glucose-6-phosphate, 3 ) microsome mix (microsomes diluted 1:2 in phosphate buffer 0.01 M pH 7.4; 1.2 nmol of cyto- chrome P45O/mg of protein, 4) microsomes mix without NADP or glucose-6-phos- phate, or 5) SOD and catalase. The chemical was incubated during 2 hr of the treatment period. The activities of SOD and catalase used were 74.6 and 265 U/flask, respec-
232 Duarte Silva et al.
tively, which correspond to the activities of SOD and catalase present in 500 pl of S9 Mix. Experiments with catalase (265 U) and SOD (74.6 U) alone were also per- formed; 15 pl of DMSO was used in control experiments, either with 265 U of catalase and 74.6 U of SOD or 500 pl of SlOO Mix. One unit of SOD is defined as the amount of protein that inhibits the autooxidation of pyrogallol by 50%, followed at 420 nm. One enzyme unit of catalase is the amount of protein that decomposes 1 pmol of H,OJmin, followed spectrophotometrically at 240 nm. SlOO Mix was pre- pared as S9 Mix [21]. Microsomes mix was prepared as S9 Mix but 28 U of glu- cose-6-phosphate dehydrogenase was added per 500 p1 of mix.
MN Assay Twenty hour cultures (approximately 3 x lo5 cells), grown in microwell plates
with a glass slide on the bottom, were washed with Ham’s F-10 medium reconsti- tuted in phosphate buffer 0.01 M pH 7.4 and grown in 3 ml of this medium for 2 hr in the presence of kaempferol (dissolved in DMSO, of which the final concentration did not exceed 56 mM). When metabolic activation was required, 300 p1 of S9 Mix, 10% (v/v), and 2.7 ml of medium were used. After the treatment, cells were washed with culture medium and grown for another 17 hr in the presence of cytochalasin B (6 pg/ml). Cells were then washed 5 times with phosphate buffered saline (PBS) (4°C) fixed with cold methanol for 5 min, washed and fixed again for 20 min, and then stained with Giemsa (4% in phosphate buffer 0.01 M pH 6.8) for 15 min. Two independent experiments were carried out with each test compound and 1,000 binucleated cells were scored for each dose.
HPLC Analysis Kaempferol (69.6 pM) was incubated with either 1) 500 p1 of rat liver S9 mix,
2) 500 p1 of microsomes mix with and without cofactors, or 3) 500 p1 of S100, at 37°C for different times. The reaction was stopped by the addition of H3P04 in metha- nol 0.1 M as described by Vrijsen et al. [23]. The mixture was centrifuged and the supernatant was evaporated to dryness. The residue was reconstituted in 75 pl DMSO and then diluted 1:3 in acetic acid lo%, filtered through 0.2 pm Nters (Anatop 10, Merck) and analyzed in a Merck-Hitachi L-6200 HPLC apparatus with a diode array Merck- Hitachi L-3000 and a RP-18 column. We have applied a linear gradient of 10% acetic acid (A) in aqueous solution and acetonitrile (B) from 15 to 90% acetonitrile in 20 min. The flow was maintained at 0.7 ml/min and the detection followed at 360 nm.
RESULTS
The results on the induction of CAs by kaempferol in V79 cells with and with- out S9 Mix are shown in Table I. There is a dose-dependent induction of CAs in V79 cells, but this induction significantly increases in the presence of S9 Mix ( P < 0.05 for the doses of 52.2, 104.4, and 139.2 pM).
The results on the induction of MN by kaempferol in V79 cells with and with- out S9 are shown in Table 11. There is a dose-dependent induction of MN in V79 cells, but this induction significantly increases in the presence of S9 Mix ( P < 0.01 for 17.4 pM; P < 0.001 for 52.2 and 104.4 pM).
The results on the induction of CAs by 104.4 pM kaempferol in V79 cells in the presence of microsomes mix, microsomes mix without cofactors, S9 Mix with-
TA
BL
E I.
Ind
uctio
n of
CA
s by
Kae
mpf
erol
in V
79 C
ells
With
out a
nd W
ith S
9 M
ixT
Tota
l To
tal
% A
berr
ant
% A
berr
ant
aber
ratio
ns
aber
ratio
ns
cells
ce
lls
incl
udin
g ex
clud
ing
incl
udin
g ex
clud
ing
Mito
tic
gaps
ga
ps
gaps
ga
ps
inde
x (%
) D
ose
(pM
) C
tg
Ctb
In
t C
hg
Chb
D
ic
Oth
ers
>lo
a
0 Ex
p 1
3 1
01
0
0 0
0 5
1 5
1 6.
4 E
xp2
3 1
00
0
0 0
0 4
1 4
1 12
.3
v79
17.4
E
xpl
2 0
0 0
0 0
0 0
2 0
2 0
Exp
2 2
1 0
0 0
0 0
0 3
1 3
1 52
.2
Exp
1 1.5
3
1 1
0 1
0 0
21
5 18
5
Exp
2 18
4
1 1
0 1
0 0
25
6 21
4
104.
4 E
xpl
15
8 9
2 0
0 0
0 34
17
25
11
E
xp2
19
7 3
1 0
1 1
0 32
12
22
10
Exp
2 13
7
5 1
0 0
1 0
27
13
13
13
139.
2 E
xpl
22
13
12
1 0
0 0
0 48
25
30
15
MM
C
Exp
l 5
7 8
1 0
2 0
2 23
17
20
15
3
Exp
2 V79
With
S9 M
ix
0 E
xpl
6 0
0 0
0 0
0 0
6 0
5 0
Exp
2 5
0 0
0 0
0 0
0 5
0 5
0 17
.4
Exp
1 5
10
0 0
0
0 0
6 1
6 1
Exp
2 3
0 0
0 0
1 0
0 4
1 4
1 52
.2
Exp
l 9
13
0 2
0 0
0 0
24
13
16
10*
Exp
2 5
9 0
1 0
0 0
0 15
9
15
9
139.
2 E
xpl
21
13
4 1
2 2
1 0
44
22
36
21*
4
104.
4 Ex
p 1
21
8 3
2 2
7 0
0 43
20
34
16
* E
xp2
5 6
3 0
1 8
0 0
23
18
20
17
Exp
2 19
15
4
1 1
3 0
0 43
23
34
20
3.
8 CP
Ex
p 1
13
16
5 2
0 4
0 0
40
25
27
19
'Ctg
= c
hrom
atid
gap
; Ctb
= c
hrom
atid
bre
ak; I
nt =
inte
rcha
nge;
Chg
= c
hrom
osom
e gap
: Chb
= c
hrom
osom
e bre
ak; D
ic =
dic
entri
cs; M
MC
= m
itom
ycin
C; C
P =
cy
clop
hosp
harn
ide.
"M
ultia
berr
ant c
ells
. *P
< 0.
05 c
ompa
red
with
the
sam
e do
ses
in V
79 c
ells
.
21.5
E
xp2
6 8
18
0 1
4 1
1 38
32
22
20
1.9
3.3
10.6
6.
6
234 Duarte Silva et al.
TABLE 11. Induction of MN in V79 Cells by Kaempferol Without and With S9 Mix’
Dose(pM) V79 % CB %Poly %MNCB % MonoMN %Met
0 Exp 1 98.2 8 18 - 0.1 Exp 2 98.1 4.4 19 0.1
52.2 Exp 1 95.7 11.9 43 0.1 Exp 2 95.8 5.2 42 0.1
104.4 Exp 1 93.5 16.2 65 0.1 Exp 2 94.1 3 59 0.1
-
- 17.4 Exp 1 97.1 7.2 29 -
Exp 2 97.4 5.5 26 - - -
-
-
-
Dose (pM) V79 S9 % CB %Poly %MNCB %MonoMN %Met
0 Exp 1 Exp 2 91.6 3.2 24 0.3 0.3
17.4 Exp I 94.2 5.9 58* 0.7 0.2 Exp 2 93.9 5 61 1.2
52.2 Exp I 91.2 6 88** 0.2 -
Exp 2 91.1 3.4 89 0.8
Exp 2 90 3.8 100 0.7 0. I
-
- 104.4 Exp 1 89.8 6.1 102** 0.6
‘CB = % binucleated cells; % Poly = % polynucelated cells: % MNCB = % binucleated cells with MN; % Mono MN = % mononucleated cells with MN; % Met = % metaphases. * P < 0.01 compared with the same dose in V79 cells. **P < 0.001 compared with the same dose in V79 cells.
out cofactors, S 100, SOD and catalase are shown in Table 111. S9 Mix without cofac- tors, S100, and SOD and catalase have no influence on the induction of CAs. When comparing the induction of CAs in the presence of microsome mix with and without cofactors it is observed that there is a significant increase when cofactors are present ( P < 0.05).
The results on the pH-dependent induction of CAs by 104.4 pM kaempferol are shown in Table IV. Kaempferol induces CAs at all of the pHs tested and there are no significant differences in this induction when the pH of the medium is raised from 6.0 to 8.0.
The results obtained using HPLC after incubation of kaempferol with microsome mix are shown in Figure 1. There is a time-dependent biotransformation of kaempferol into quercetin in the presence of either microsome mix (Fig. la-c) or S9 Mix (not shown). This transformation does not occur if kaempferol is incubated with microsome mix without cofactors (Fig. Id), SlOO, or with S9 Mix without cofactors (not shown). The identification of quercetin was carried out by comparison of retention times and ultraviolet( UV) spectra between the samples and standards.
DISCUSSION
Kaempferol is a flavonoid widely distributed in the plant kingdom and is present in the edible portions of the majority of food plants [4]. Normally it appears in the form of glycosides which are not mutagenic [lo], but these glycosides can be hydrolyzed by bacterial glycosidases present in the lower gut giving rise to the free mutagenic aglicone [24]. It is desirable to obtain more data on the mechanisms of mutagenicity of kaempferol in order to define its potential to produce genetic damage.
TA
BL
E 11
1. In
duct
ion
of C
As
by K
aem
pfer
ol in
V79
Cel
ls in
the
Pres
ence
of V
ario
us L
iver
Ext
ract
s, W
ith o
r W
ithou
t C
ofac
tors
, and
in th
e Pr
esen
ce o
f the
E
nzym
es C
atal
ase
(Cat
) and
SO
Dt
Tota
l To
tal
% A
berr
ant
% A
berr
antt
aber
ratio
ns
aber
ratio
ns
cells
ce
lls
incl
udin
g ex
clud
ing
incl
udin
g ex
clud
ing
Trea
tmen
t D
ose
(pM
) C
tg
Ctb
In
t C
hg
Chb
D
ic
Oth
ers
>lo
” ga
ps
gaps
ga
ps
gaps
104.
4 Ex
p 1
13
7 1
1 1
0 0
0 23
9
18
9 E
xp2
17
11
3 1
0 2
0 0
34
17
23
12
Exp
3 23
6
4 0
0 2
1 0
36
13
28
11
Exp
4 23
9
5 0
1 1
0 0
39
16
28
9
Exp
2 14
13
9
1 1
4 0
0 41
27
27
20
SlO
O
0 Ex
p 1
5 0
1 0
0 0
0 1
6 1
7 2
Exp
2 6
1 0
0 1
0 1
0 9
2 5
2 10
4.4
Exp
1
14
17
7 2
0 0
1 0
30
14
22
12
Exp
2 14
11
4
1 0
2 0
0 32
17
22
12
S9
-cof
0
Exp
1
0 0
0 0
0 0
0 0
0 0
0 0
Exp
2 1
0 0
0 0
0 0
0 1
0 1
0 10
4.4
Exp
1 13
5
0 1
3 0
0 0
22
8 20
8
SOD
+Cat
0
Exp
1 1
0 0
0 0
0 0
0 1
0 1
0 E
xp2
1 0
0 1
0 0
0 0
2 0
2 0
Exp
2 12
6
4 4
0 1
0 0
27
11
23
10
s9
104.
4 Ex
p 1
17
10
1 4
6 4
0 0
42
21
33
19
Exp
3 16
11
1
2 1
3 0
0 34
16
28
16
E
xp4
28
14
4 2
0 1
0 0
49
19
37
17
Exp
2 12
6
4 0
1 2
0 0
25
13
23
13
104.
4 E
xp
l 13
6
5 0
1 2
0 0
27
14
22
13
MIC
10
4.4
Exp
1 6
6 0
1 7
1 0
0 21
14
18
13
E
xp2
12
10
0 3
2 3
0 1
30
15
25
15
Exp
3 16
10
2
4 1
5 0
0 38
18
28
13
M
IC-c
of
104.
4 E
xp 1
4
3 2
3 1
1 0
0 14
7
13
7*
Ex
~2
8
7 0
1 2
1 0
0 19
10
17
10
*
‘Ctg
= c
hrom
atid
gap
; Ctb
= c
hrom
atid
bre
ak; I
nt =
inte
rcha
nge;
Chg
= c
hrom
osom
e ga
p; C
hb =
chr
omos
ome
brea
k; D
ic =
dic
entr
ics.
“M
ultia
berr
ant c
ells
. *P
< 0
.05
com
pare
d to
V79
cel
ls in
the
pres
ence
of
mic
roso
mes
(M
IC) m
ix.
TABL
E IV
. In
duct
ion
of C
As b
y K
aem
pfer
ol in
V79
Cel
ls a
t Diff
eren
t pH
Val
ues'
Trea
tmen
t D
ose
(pM
) C
tg
Ctg
In
t C
hg
Chb
pH 6
.0
0 Ex
p 1
3 2
0 0
0 E
xp2
3 0
0 0
0 10
4.4
Exp
1 14
3
4 3
0 E
xp2
12
6 2
4 0
pH 7
.4
0 Ex
p 1
0 0
0 0
0 E
xp2
0 0
0 0
0 10
4.4
Exp
l 17
11
3
1 0
Exp
2 23
6
4 0
0 pH
8.0
0 Ex
p 1
2 0
0 1
0 E
xp2
1 1
0 0
0 10
4.4
Exp
1 12
1
1 2
4 E
XD
~ 13
6
3 0
0
Tota
l To
tal
% A
berr
ant
% A
berr
antt
aber
ratio
ns
aber
ratio
ns
cells
ce
lls
incl
udin
g ex
clud
ing
incl
udin
g ex
clud
ing
Dic
O
ther
s >
loa
gaps
ga
ps
gaps
ga
ps
0 0
0 5
2 5
2 2
0 0
5 2
5 2
0 1
0 25
8
19
8 0
1 0
27
10
22
8 0
0 0
0 0
0 0
0 0
0 0
0 0
0 2
0 0
34
17
23
12
2 1
0 36
13
28
11
0
0 0
3 0
3 0
0 0
0 2
1 2
1 1
1 0
22
8 20
8
0 3
0 25
12
18
11
'Ctg
= ch
rom
atid
gap
; Ctb
= c
hrom
atid
bre
ak; I
nt =
inte
rcha
nge;
Chg
= ch
rom
osom
e ga
p; C
hb =
chro
mos
ome
brea
k; D
ic =
dic
entri
cs.
"Mul
tiabe
rran
t cel
ls.
Mutagenicity of Kaempferol in V79 Cells 237
In the present work we show that kaempferol induces CAs in V79 cells in a dose-dependent manner (Table I). The same kind of results are obtained when the induction of MN is used as the genetic endpoint (Table II), which is to be expected given the fact that these two endpoints are related.
Given the known prooxidant activity of flavonoids, under conditions where they are able to autooxidize giving rise to the superoxide anion and consequently hydro- gen peroxide, as widely described [13,19,25,26], we decided to ascertain the role of radicalar species on the mutagenicity of kaempferol. Kaempferol itself has been shown to induce nuclear DNA damage and lipid peroxidation by a mechanism dependent on the formation of oxygen radicals [25]. The induction of CAs at different pHs (Table IV) and in the presence of the antioxidant enzymes SOD and catalase (Table 111) is comparable to the induction by kaempferol, which suggests that a radicalar species are not involved in the genotoxic action of kaempferol.
Given these facts it seems appropriate to conclude that kaempferol, in these conditions, is able to enter the cell and reach the DNA and cause genetic lesions. This is not surprising since flavonoids are known to bind lo DNA [27] and may be able to induce genetic lesions.
In the presence of S9 Mix the induction of both CAs and MN by kaempferol is increased, when compared to its absence. This suggests a metabolization pathway of kaempferol to a more potent genotoxicant. To further investigate this hypothesis we assessed the induction of CAs in the presence of S9 Mix without NADPH, microsomes with NADPH, microsomes without NADPH and S100, the cytosolic fraction of S9 Mix. The results obtained (Table 111) suggest that cytochromes P450 play a role in the metabolization of kaempferol. It must be noted that the response with microsome mix is lower than with S9 Mix, but these situations cannot easily be compared. It is relevant, however, to compare the results obtained with S9 Mix with and without cofactors and with microsome mix with and without cofactors. The presence of co- factors increases the induction of CAs. These results corroborate the results obtained by Maruta et al. [17] which showed that S9 Mix was able 1;o metabolize kaempferol to a product that could induce hprt mutations in V79 cells. The fact that they did not see any mutations with kaempferol alone may be due to the different endpoint studied.
In order to identify the product into which kaempferol is transformed by cyto- chromes P450 we performed time-dependent incubations with S9 Mix and microsome mix (Fig. 1) and observed, as followed by HPLC, a time-dependent transformation of kaempferol into quercetin in both conditions. Incubations with S9 Mix and mi- crosome mix without cofactors and with SlOO served as negative controls. Brown and Dietrich [ 101 have suggested that kaempferol could be transformed into querce- tin by a mechanism dependent on cytochromes P450. In addition, Zhu et al. [28], when referring to the paper by Maruta et al. [17], have suggested that kaempferol could be transformed into a product having a catechol moiety by S9 Mix. We show here that the suggestions of those authors were correct and that cytochromes P450 are able to transform kaempferol into quercetin.
In view of all these results it seems fair to conclude that cytochromes P450 are able to transform kaempferol into quercetin and that quercetin is responsible for the increased genotoxicity of kaempferol when metabolic activation is used. Quercetin has been shown to induce CAs in V79 cells [13] by a mechanism dependent on the production of oxygen radicals outside the cell. In this case oxygen radicals do not seem to play a role but we must bear in mind that the conditions studied are differ-
238 Duarte Silva et al.
0 min
a
Kaempferol
10 11 12 13 14 15 16 17 18 19 20
Time (min.)
60 min
b I T I
10 11 12 13 14 15 16 17 18 19 20
Time (min.)
Fig. 1 . crosomes with cofactors (a-c) and without cofactors (d).
Time-dependent HPLC analysis of the biotransformation of kaempferol to quercetin by mi-
Mutagenicity of Kaempferol in V79 Cells 239
120 rnin
C
Quercetin I
7 - I
10 11 12 13 14 15 16 11 7 18 19 20 Time (min.)
Figure lc.
120 min without cofactors
Kaempferol
10 11 12 13 14 15 16 -17 18 19 20
Time (min.)
Figure Id.
240 Duarte Silva et al.
ent, namely that the only quercetin present is generated inside microsomes and this can affect the permeability of the cell to quercetin and the access of the antioxidant enzymes to quercetin.
It can be concluded that kaempferol is biotransformed to quercetin by cyto- chromes P450 and the latter seems to contribute to the genotoxicity of kaempferol in the presence of metabolic activation systems. ROS do not seem to play a role in the genotoxicity of kaempferol. Work is in progress to identify the cytochrome(s) P450 responsible for its biotransformation using genetically engineered cells expressing different cytochromes P450, and also specific inhibitors for these enzymes.
ACKNOWLEDGMENTS
Our current research is supported by the European Commission. We also ac- knowledge the collaboration of the Centre for Malaria and Other Tropical Diseases- UNL. J.G. is supported by a postdoctoral fellowship and I.D.S. is supported by a doctoral fellowship from Junta Nacional de Investiga@o Cientifica e Tecnol6gica. We are grateful to Mr. Romiio from the Gulbenkian Institute of Sciences for his excellent technical assistance.
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