Metabolism of genistein by rat and human cytochrome P450s

Article abstract of DOI:10.1021/tx9802320

The metabolism of genistein (4',5,7-trihydroxyisoflavone), a phytoestrogen derived from soy products, was investigated using rat and human liver microsomes and recombinant human cytochrome P450 enzymes. Metabolism of genistein by microsomes obtained from rats treated with pyridine, phenobarbital, β-naphthoflavone, isosafrole, pregnenolone-16α-carbonitrile, or 3-methylcholanthrene resulted in very different product profiles consisting of five different NADPH- and time-dependent metabolites as observed by HPLC reverse-phase analysis at 260 nm. The metabolism of genistein was also investigated with recombinant human cytochrome P450 1A1, 1A2, 1B1, 2B6, 2C8, 2E1, or 3A4. P450s 1A1, 1A2, 1B1, and 2E1 metabolized genistein to form predominantly one product (peak 3) with smaller amounts of peaks 1 and 2. P450 3A4 produced two different products (peaks 4 and 5). Product peaks 1-3 eluted off the HPLC column prior to the parent compound genistein, and the UV/vis spectra, GC/MS, and ESI/MS/MS analyses support the conclusion that these products result from hydroxylation of genistein. The product peak 3 has been identified by tandem mass spectrometry as 3',4',5,7- tetrahydroxyisoflavone, also known as orobol, and peaks 1 and 2 appear to be hydroxylated at position 6 or 8.

Full text of DOI:10.1021/tx9802320

6
10
Chem. Res. Toxicol. 1999, 12, 610-616
Meta bolism of Gen istein by Ra t a n d Hu m a n
Cytoch r om e P 450s
,
†
‡
§
Elizabeth S. Roberts-Kirchhoff,* J an R. Crowley, Paul F. Hollenberg, and
|
Hyesook Kim
Department of Chemistry and Biochemistry, University of Detroit Mercy, Detroit, Michigan 48219,
Medical Mass Spectrometry Facility, Washington University, St. Louis, Missouri 63110,
Department of Pharmacology, University of Michigan, Ann Arbor, Michigan 48109,
Oxford Biomedical Research, Rochester Hills, Michigan 48309, and Institute of Chemical
Toxicology, Wayne State University, Detroit, Michigan 48201
Received October 16, 1998
The metabolism of genistein (4′,5,7-trihydroxyisoflavone), a phytoestrogen derived from soy
products, was investigated using rat and human liver microsomes and recombinant human
cytochrome P450 enzymes. Metabolism of genistein by microsomes obtained from rats treated
with pyridine, phenobarbital, â-naphthoflavone, isosafrole, pregnenolone-16R-carbonitrile, or
3
-methylcholanthrene resulted in very different product profiles consisting of five different
NADPH- and time-dependent metabolites as observed by HPLC reverse-phase analysis at 260
nm. The metabolism of genistein was also investigated with recombinant human cytochrome
P450 1A1, 1A2, 1B1, 2B6, 2C8, 2E1, or 3A4. P450s 1A1, 1A2, 1B1, and 2E1 metabolized
genistein to form predominantly one product (peak 3) with smaller amounts of peaks 1 and 2.
P450 3A4 produced two different products (peaks 4 and 5). Product peaks 1-3 eluted off the
HPLC column prior to the parent compound genistein, and the UV/vis spectra, GC/MS, and
ESI/MS/MS analyses support the conclusion that these products result from hydroxylation of
genistein. The product peak 3 has been identified by tandem mass spectrometry as 3′,4′,5,7-
tetrahydroxyisoflavone, also known as orobol, and peaks 1 and 2 appear to be hydroxylated at
position 6 or 8.
In tr od u ction
Dietary intake of soy is associated with lower incidence
rates of hormonally dependent and independent cancers
1). Soy contains large amounts of the isoflavones,
genistein (4′,5,7-trihydroxyisoflavone) and daidzein (4′,7-
dihydroxyisoflavone) (2). Long-term systemic exposure to
isoflavonoids, including genistein, may prevent the de-
velopment of breast cancer and delay progression of
latent prostatic carcinoma (3-5). Genistein is also known
to suppress the growth of solid tumors (6, 7) and various
forms of leukemia (8-10). While a specific mechanism
of action has not been identified, genistein is known to
inhibit tyrosine specific protein kinases (9, 10), DNA
topisomerase II (9, 11), epidermal growth factor-induced
phosphatidylinositol turnover (12), S6 kinase activation
6′′-O-acetylglucoside, and the 6′′-O-malonylglucoside as
reviewed by Reinli and Block (18). The hydrolysis of the
glycosides occurs in the gastrointestinal tract. The ab-
sorbed phytoestrogen metabolites undergo enterohepatic
circulation and may be excreted in the bile, deconjugated
by intestinal flora, reabsorbed, reconjugated by the liver,
and excreted in the urine (19, 20). Isoflavones have been
found in human fluids, including urine, plasma, and
breast milk (21, 22). Concentrations of the different
phytoestrogen metabolites in the body vary widely and
depend on intestinal flora, antibiotic use, bowel disease,
gender, and genetic factors as reviewed by Murkies et
al. (19) and Aldercreutz and Mazur (23).
(
The cytochrome P450 superfamily of genes encodes a
variety of proteins found in nearly every tissue, and these
enzymes catalyze the metabolism of both endogenous
compounds and xenobiotics (24). P450s also play a
significant role in the metabolism of some procarcinogens
to carcinogens (24). P450s are known to hydroxylate
estradiol and also play a role in the biosynthesis of
isoflavones in cell cultures of Pueraria lobata (25) and
the metabolism of isoflavones in rats (26-28). Since
P450s are abundant in the liver and small intestine (29)
where much of the metabolism of isoflavones is reported
to occur (19, 20), P450s may play a role in the metabolism
of isoflavones, including genistein. In this study, we
investigate the metabolism of genistein in liver mi-
crosomes from rats treated with various P450-inducing
agents, including pyridine, phenobarbital, â-naphthofla-
vone, isosafrole, pregnenolone-16R-carbonitrile, or 3-me-
(
13), and angiogenesis (14). In addition, genistein also
may exert its effect during the administration of a
carcinogen to the animal by decreasing the activity of the
enzymes that activate the carcinogen (15).
While the metabolism of isoflavones is poorly under-
stood in humans, the metabolism of several isoflavones
has been characterized in sheep (16, 17). In plants,
genistein exists as the aglycone, the 7-O-glucoside, the
*
To whom correspondence should be addressed: Department of
Chemistry and Biochemistry, University of Detroit Mercy, P.O. Box
1
9900, Detroit, MI 48219-0900.
†
University of Detroit Mercy.
Washington University.
University of Michigan.
Oxford Biomedical Research and Wayne State University.
‡
§
|
1
0.1021/tx9802320 CCC: $18.00 © 1999 American Chemical Society
Published on Web 06/04/1999

Metabolism of Genistein by Cytochrome P450
Chem. Res. Toxicol., Vol. 12, No. 7, 1999 611
thylcholanthrene. We also investigate the metabolism of
genistein in several human liver microsomal samples and
with recombinant human P450s. The metabolites were
characterized by HPLC, UV/vis spectroscopy, GC/MS,
and ESI/MS/MS.
Exp er im en ta l P r oced u r es
Ma ter ia ls. Genistein and NADPH were from Sigma Chemi-
cal Co. (St. Louis, MO). Supersomes, microsomes obtained from
baculovirus insect cells containing cDNA-expressed reductase,
5
and P450s 1A1, 1A2, 1B1, 2B6, 2C8, 2E1 (with cytochrome b ),
and 3A4 were from Gentest Corp. (Woburn, MA). Human liver
microsomes were a gift from F. P. Guengerich (Vanderbilt
University, Nashville, TN). Rat liver microsomes were prepared
according to the method of Saito and Strobel (30) from the livers
of male, 10-week-old Fisher 344 rats (Charles River Laborato-
ries, Portage, MI) given 100 mg of pyridine/kg (ip for 3 days),
8
0 mg of â-naphthoflavone/kg (ip for 3 days), 0.1% phenobarbital
in the drinking water for 12 days, 50 mg of pregnenolone-16R-
carbonitrile/kg (ip for 4 days), 150 mg of isosafrole/kg (ip for 4
days), or 25 mg of 3-methylcholanthrene/kg (ip for 3 days).
Meta bolism of Gen istein . Genistein was incubated with
microsomes (0.5 mg of rat or human liver microsomal protein
or 0.05-0.1 nmol of baculovirus-expressed P450) for 0-30 min
at 37 °C with 1.0 mM NADPH in 50 mM potassium phosphate
buffer (pH 7.4) in a final volume of 1.0 mL. The incubations
2
containing P450 2E1 or 3A4 also contained 3 mM MgCl .
F igu r e 1. Representative reverse-phase HPLC profiles of ethyl
acetate extracts of incubations containing rat liver microsomes
and genistein. Each reaction mixture contained 150 µM genistein
and 0.5 mg of microsomal protein from untreated rats (control)
or rats treated with pyridine (Pyr) or phenobarbital (PB).
Incubations and HPLC analyses were as described in Experi-
mental Procedures. Genistein eluted at 21 min, and product
peaks 1-5 are shown.
Genistein and its metabolites were isolated from incubations
that had been quenched with 50 µL of 30% phosphoric acid and
then extracted with ethyl acetate. The organic layer was dried
under N , and the residue dissolved in HPLC buffer. These
2
fractions were analyzed on a Gilson HPLC system at 260 nm
on a C18 reverse-phase column with solvent A (0.1% trifluoro-
3 2
acetic acid) and solvent B (95% CH CN/5% H O/0.1% trifluo-
roacetic acid). A linear gradient of 5 to 60% B over the course
of 30 min separated the components. Quantitation of metabolites
was based on a standard curve using known amounts of
genistein since the proposed metabolites are not commercially
available. This is a good approximation of the amount of
products formed since (1) the peak maxima of the products are
within 7 nm of the peak maximum of genistein (as determined
by HPLC analysis with diode array detection), (2) they are
hydroxylated metabolites of genistein (as determined by mass
spectrometry), and (3) several isoflavones closely related to
genistein, including daidzein, formononetin, and biochanin A,
have extinction coefficients that differ at the most by a factor
of 1.5 (31).
4
in 200 µL of MeOH/10 mM NH OAC (50:50) and were sprayed
into the ion source by continuous infusion at a rate of 5 µL/
min. The temperature of the heated capillary was 150 °C, and
2
the sheath gas (N ) flow rate was 50 mL/min. The spray voltage
was 4.5 kV. Product ion spectra were obtained using a collision
energy setting of 27% of the resonance RF energy. Helium was
introduced to an estimated pressure of 1 mTorr for improving
trapping efficiency.
Resu lts
When genistein was incubated with rat liver mi-
crosomes in the presence of NADPH, several product
peaks were observed by HPLC analysis at 260 nm (Figure
1). Five different products were observed when genistein
was metabolized by microsomes from pyridine- or phe-
nobarbital-induced rats. These products were not ob-
served when NADPH was omitted from the incubation
mixtures. The formation of these products with mi-
crosomes from pyridine or phenobarbital-treated rats was
time-dependent (data not shown). The formation was
somewhat linear early in the incubation but appeared
to deviate significantly from linearity at later incubation
times (15-30 min). If microsomes from rats treated with
other P450-inducing agents were used to metabolize
genistein, the results in Figure 2 were obtained. Induc-
tion with pyridine, 3-methylcholanthrene, or â-naph-
thoflavone favored the production of products 1-3, while
induction with phenobarbital or pregnenolone-16R-car-
bonitrile favored the production of peaks 4 and 5. Thus,
the metabolism of genistein is NADPH- and time-
dependent and results in the production of five different
metabolites. The relative amount of each metabolite is
dependent upon the inducing agent that was used.
Ma ss Sp ectr om etr y. GC/MS and ESI/MS/MS were per-
formed at the Medical Mass Spectrometry Facility (Washington
University). Genistein and its metabolites were separated by
HPLC and extracted into ethyl acetate. For GC/MS analysis,
isoflavones in the dried extracts were converted to their tri-
1
methylsilyl (TMS) ether derivatives by heating for 15 min at
6
5 °C with BSTFA. The gas chromatograph was a Hewlett-
Packard model 5890, and the column that was used was a DB1
12 m, 0.2 mm i.d., 0.33 mm methyl silicone film coating; P. J .
(
Cobert, St. Louis, MO). The injector and detector temperatures
were set to 250 °C. The isothermal column temperature was
2
5
60 °C. EI/MS spectra were acquired on a Hewlett-Packard
988A mass spectrometer (Palo Alto, CA). The emission current
was set to 300 mA. The electron energy was 70 eV, and the
source temperature was 200 °C. The source pressure was
maintained at about 0.5 Torr with methane as the reagent gas.
ESI/MS/MS anaylses were performed on a Finnigan quad-
rupole ion trap (LCQ) mass spectrometer equipped with a
Finnigan ESI source operated in the positive ion mode with the
automatic gain control active. The dried samples were dissolved
1
Abbreviations: TMS, trimethylsilyl; BSTFA, N,O-bis(trimethyl-
silyl)trifluoroacetamide with 1% trimethylchlorosilane; CAD, collision-
activated dissociation; ESI, electrospray ionization.

6
12 Chem. Res. Toxicol., Vol. 12, No. 7, 1999
Roberts-Kirchhoff et al.
F igu r e 2. Metabolism of genistein by liver microsomes from rats treated with various inducing agents. Each reaction mixture
contained 0.5 mg of protein, 80 µM genistein, and NADPH, and the incubation time was 10 min. Incubations and HPLC analysis
were as described in Experimental Procedures. Quantitation is based on a standard curve using known amounts of genistein. Rats
were untreated (Con) or treated with phenobarbital (Pb), pyridine (Pyr), isosafrole (Iso), 3-methylcholanthrene (3MC), â-naphthoflavone
(bNF), or pregnenolone-16R-carbonitrile (PCN). Mean values ( standard deviations from three independent experiments are plotted.
The kinetic constants for the formation of peaks 1-3
Ta ble 1. Kin etic Con sta n ts for th e F or m a tion of P ea k s
1
-3 fr om Gen istein by Micr osom es fr om
from genistein by microsomes from pyridine-treated rats
P yr id in e-Tr ea ted Ra tsa
are given in Table 1. From Eadie-Hofstee plots of the
peak
Km (µM)
30
Vmaxb (nmol min-1 mg of protein-1
)
data, high and low K
m
values were determined for the
metabolism of genistein to peak 3. This could be ex-
plained if microsomes from pyridine-treated rats con-
1
2
3
3
0.89
1.2
1.4
35
16 (low Km)
104 (high Km)
tained a high- and low-K
m
P450 responsible for the
4.4
metabolism of genistein to peak 3.
a
The results from the metabolism of genistein by three
different human liver microsomal samples are shown in
Figure 3. Each microsomal sample exhibits a different
product profile. Each of the human liver microsomal
samples has been analyzed for various P450 marker
activities; the sample designated as 100 has high 2E1-
Incubation mixtures contained 2-200 µM genistein, and the
incubation time was 10 min. Other incubation conditions were as
described in Experimental Procedures. Kinetic constants were
obtained from Eadie-Hofstee plots of the data. b The values are
approximate and based on quantitation with a standard curve
using known amounts of genistein as described in Experimental
Procedures.

Metabolism of Genistein by Cytochrome P450
Chem. Res. Toxicol., Vol. 12, No. 7, 1999 613
F igu r e 3. Genistein metabolism by human liver microsomes.
Experimental conditions were as described in the legend of
Figure 2 and in Experimental Procedures.
F igu r e 5. GC/MS analysis of the TMS derivative of genistein
and peak 3. Peak 3 was isolated from an incubation containing
microsomes from pyridine-induced rats.
3
is a hydroxylated metabolite of genistein. The TMS
derivative of genistein has a molecular weight of 486, and
+
while the M ion is present at an intensity of <1%, the
+
M - 15 ion is strong and can be used to determine the
molecular weight. The loss of an OTMS group results in
the ion at m/z 399. Peak 3 has a strong ion at m/z 559
which corresponds to the addition of an OTMS group. The
ions at m/z 471 and 399 result from losses of additional
F igu r e 4. Genistein metabolism by recombinant human P450s.
The incubation and HPLC conditions were as described in
Experimental Procedures. (A) Production of product peaks 1-3.
OTMS and CH
3
groups. More information about the
(B) Production of product peaks 4 and 5 by P450 3A4.
position of the hydroxyl group was obtained by the use
of electrospray ionization mass spectrometry (ESI). The
ESI/MS spectrum of peak 3 displayed in Figure 6 shows
specific chloroxazone hydroxylase activity, while sample
10 has high 3A4-specific nifedipine oxidation activity
32). Thus, as seen previously with rat liver microsomes
+
1
(
the [M + H] ion (panel A). Upon CAD, this ion is further
fragmented as shown in the spectrum of panel B. Tandem
mass spectrometry of isoflavones will fragment to give
from animals treated with different P450-inducing agents,
the genistein product profile is dependent on the P450
profile of the microsomes. Genistein was also metabolized
to various products when incubated with insect cell
microsomes containing recombinantly expressed human
P450s and reductase (Figure 4). P450s 1A1, 1A2, and, to
a lesser extent, 1B1 and 2E1 metabolized genistein to
peaks 1-3. Only P450 3A4 was found to metabolize
genistein also to give peaks 4 and 5.
The UV spectra of the product peaks were obtained
by HPLC with diode array detection (data not shown).
The UV spectrum appeared to be similar to that of
genistein for each of the five compounds with peak
maxima at 267 nm for peak 1, 270 nm for peak 2, 262
nm for peak 3, 265 nm for peak 4, and 264 nm for peak
ions derived from consecutive losses of H O and CO as
2
well as ions indicative of substituent positioning (33). The
retro Diels-Alder decomposition product, a predominate
ion in the CAD spectra of all flavonoids (Scheme 1), can
be used to determine the number of substituents on the
A ring. The mass at m/ z 153 is a retro Diels-Alder
fragment which demonstrates that there are two hy-
droxyl groups on the A ring. This ring has not been
altered from the original parent genistein. An additional
ion fragment arises from bond cleavage to give a frag-
ment containing the B ring. The fragment, m/z 161, was
only seen previously when there were 3′,4′-hydroxyl
groups on the B ring (33). The possible structure is a
+
quinone as shown in Scheme 1. The losses at MH - 18
+
+
+
5
. The GC/MS (electron impact) analysis of the TMS
(269), MH - 28 (259), MH - 18 - 28 (241), and MH
derivative of peak 3 (Figure 5) demonstrated that peak
- 28 - 28 (231) are common to hydroxy isoflavones,

6
14 Chem. Res. Toxicol., Vol. 12, No. 7, 1999
Roberts-Kirchhoff et al.
F igu r e 6. Tandem mass spectral analysis of peak 3. Experi-
mental conditions were as described in Experimental Proce-
dures. (A) Full scan spectrum of peak 3. (B) CAD tandem mass
spectrum of [M + H]+ (m/z 287.3).
F igu r e 7. Tandem mass spectral analysis of peaks 1 and 2.
Experimental conditions were as described in Experimental
Procedures. (A) Full scan spectrum of peaks 1 and 2. (B) CAD
+
tandem mass spectrum of [M + H] (m/z 287.3).
Sch em e 1. F r a gm en ta tion P a tter n s for
identity of the material was confirmed by 1H NMR
a
Isofla von es
(acetone-d
34)
6
, 600 MHz) with comparison to literature data
(
Peaks 1 and 2 were collected together in one fraction
and subjected to ESI/MS/MS analysis. Only one parent
ion is shown in Figure 7 (in panel A with an ion at m/ z
2
87.3). This also corresponds to a hydroxylated metabo-
lite of genistein. Thus, peaks 1 and 2 could be hydroxyl-
ated metabolites (hydroxylation at different positions on
the A ring) that have the same molecular weight. The
fragmentation pattern of this ion is shown in panel B
and is distinctly different from that seen with peak 3
(Figure 6B). The retro Diels-Alder fragment (m/z 169.1),
which is one of the major ions in the CAD spectra of
flavonoids, results from the A ring that has an additional
hydroxyl group at either position 6 or 8. Thus, peak 1 or
peak 2 is a tetrahydroxyisoflavone with hydroxylation at
either position 6 or 8.
a
The structure of genistein is shown when R1 is H, and the
Discu ssion
structure of the hydroxylated metabolite, orobol, is shown when
R1 is OH.
We have found that genistein metabolism in rat liver
microsomes is NADPH- and time-dependent. In addition,
prototype inducers of P450 can substantially alter the
profile of hepatic genistein metabolism in rats. From the
different metabolite patterns, it is apparent that me-
tabolism by individual P450s results in different product
profiles. The Eadie-Hofstee plot of the results of genistein
metabolism to peak 3 by microsomes from pyridine-
2
resulting from the losses of H O and CO. Thus, peak
number 3 corresponds to a hydroxylated metabolite of
genistein that has been hydroxylated on the B ring at
the 3′ position to form 3′,4′,5,7-tetrahydroxyisoflavone.
This metabolite of genistein was identical with the
aglycone of orobol-7-O-glucoside when analyzed by ESI/
MS/MS (data not shown). A sample of orobol-7-O-gluco-
side was kindly provided by E. Wollenweber (Technische
Universitaet Darmstadt, Darmstadt, Germany), and the
treated rats indicates that there is a high-K
m
and a low-
K
m
enzyme with similar turnover numbers. Thus, al-

Metabolism of Genistein by Cytochrome P450
Chem. Res. Toxicol., Vol. 12, No. 7, 1999 615
though two different P450s may produce the same
product, they appear to bind genistein with significantly
different affinities. Some general conclusions can be
drawn from the data obtained with the rat liver mi-
crosomes, human liver microsomes, and recombinant
human P450s. The results from the rat liver microsomes
indicated that peaks 1-3 were present in incubations
containing microsomes from rats treated with 3-methyl-
cholanthrene, isosafrole, and â-naphthoflavone (strong
highly induced in smokers. The protein is detected in the
lungs of smokers, and gene expression is observed in
pulmonary carcinoma cells and malignant breast cancer
(44). P450 1B1 is also an extrahepatic enzyme found in
many tissues, most notably in both normal breast tissue
and breast tumors. P450 1B1 catalyzes the hydroxylation
of 17â-estradiol at position 4, resulting in a catechol that
can directly or indirectly damage macromolecules (45)
and thus initiate cancer. Isoflavones may inhibit P450s
1A1, 1A2, and 1B1, resulting in the decreased level of
formation of hydroxylated estradiol products known to
damage macromolecules. Adlercreutz and Mazur (23)
have recently reviewed the hypothesis that the isofla-
vones play a significant inhibitory role in cancer develop-
ment in the promotional phase of the disease, and recent
evidence points to a role in the initiation stage of
carcinogenesis.
1
A inducers) (35) and pyridine (a strong 2E1 and moder-
ate 2B and 1A inducer) (35, 36). Peaks 4 and 5 were
formed in incubations containing microsomes from preg-
nenolone-16R-carbonitrile (a strong 3A1 and 3A2 inducer)
(35) and phenobarbital (a strong 2B and moderate 3A
inducer) (35). The human liver microsomal sample 110,
high in 3A4-dependent activity, produced all five peaks.
Recombinant human 3A4 produced peaks 4 and 5, while
1
A1 and 1A2 and, to a lesser extent, 1B1 and 2E1
Genistein is thought to be metabolized in the gas-
trointestinal tract and liver (19, 20) which are also sites
of abundant levels of P450. In the small intestine where
there are high levels of 3A4, extensive biotransformation
of orally administered drugs occurs during absorption
across the intestinal wall (46). Much of the metabolism
of genistein is believed to be due to microbial metabolism,
but there have been observations of individual variability
in genistein metabolism that cannot be explained solely
on the basis of microbial metabolism (47). Furthermore,
there are some unidentified metabolite peaks when urine
obtained from people on soy-based diets was analyzed by
HPLC (22). More recently, genistein was found to be
metabolized to a hydroxylated metabolite in a trans-
formed human breast cancer MCF-7 cell line (48). Thus,
the available experimental information, including the
abundance of P450s in several tissues known for genistein
metabolism, the ability of P450 to metabolize a vast
number of xenobiotics and endogenous compounds, in-
cluding estradiol and other isoflavones, the data pre-
sented herein, reports of unidentified isoflavone metabo-
lites in human urine (21, 22), and genetic individual
differences in metabolism (47), suggests that human
P450s may play important roles in genistein metabolism
in vivo. Many times, P450 metabolism results in metabo-
lites that are less biologically active and more easily
excreted than the substrates, but in this case, P450
metabolism results in a compound, orobol, that is at least
as active as genistein in several in vitro tests (41-43).
P450s may have important roles in the biochemical action
of flavonoids such as gensitein by producing active
metabolites. In addition, the metabolism of isoflavonoids
by P450 may affect the metabolism of other P450
substrates, including procarcinogens.
produced peaks 1-3. More experiments are currently
underway to definitely attribute a particular metabolite
to a particular P450 by using inhibitory antibodies to
specific P450s, a bank of 18 human liver microsomes
typed for various P450-dependent activities, and specific
inhibitors of P450 isozymes. From our results, it is
apparent that (1) of the P450s tested 1A1 and 1A2 have
the highest activities for genistein metabolism, (2) dif-
ferent P450s result in different product profiles when
metabolizing genistein, and (3) P450s 1A1, 1A2, 1B1, and
2
E1 produce peaks 1-3 while only 3A4 metabolizes
genistein to peaks 4 and 5.
In this study, peak 3 was identified by GC/MS and ESI/
MS/MS analyses as a hydroxylated metabolite of genistein
(
hydroxylated on the B ring at position 3′). Peak 1 and/
or peak 2 may be a metabolite hydroxylated at positions
or 8 on the A ring. Peak 3, the 3′,4′,5,7-tetrahydroxy-
6
isoflavone, is also known as orobol and has previously
been isolated as a glucoside from a plant, Orobus tubero-
sus (37), and a fungal strain, Tritirachium sp. F3707 (38).
O. tuberosus is a herb belonging to the family Legumi-
nosae, and is now usually included in the genus Lathyrus
(
39). In Europe, the plants are sometimes cultivated for
their tubers, which have an agreeable, pleasant taste
40). Orobol has been reported to induce topoisomerase
(
II-dependent DNA cleavage to the same extent as
genistein (41), inhibit dihydroxyphenylalanine decar-
boxylase with an IC50 similar to that of genistein (42),
increase the level of cytoplasmic free calcium in isolated
rat hepatocytes (43), inhibit 15-lipoxygenase (38), and
inhibit phosphatidylinositol turnover and EGF receptor
tyrosine specific kinase in cell cultures to the same extent
as genistein (12).
In rats, P450s are reported to catalyze the transforma-
tion of the isoflavone galangin (3,5,7-trihydroxyisofla-
vone) to kaempferol (4′,3,5,7-tetrahydroxyisoflavone) and
finally to quercetin (3′,4′,3,5,7-pentahydroxyisoflavone)
Ack n ow led gm en t. We thank Dr. F. P. Guengerich
Vanderbilt University) for the gift of the human liver
(
microsomes and Dr. Yoichi Osawa (The University of
Michigan) for his assistance and the use of his Waters
HPLC system with diode array detection. We are grateful
to Dr. E. Wollenweber (Technische Universitaet Darm-
stadt) for a sample of orobol-7-O-glucoside and to Dr. J .
F. Stevens (Oregon State University, Corvallis, OR) for
NMR data. Supported in part by NIH Grants CA 16954
(26, 27). 1A1 is reported to hydroxylate kaempferol on
the B ring at the 3′ position to yield quercetin (27), while
an unidentified P450 is responsible for the first hydroxyl-
ation (27). Similarly, we have evidence that P450s 1A1,
1
A2, and 1B1 hydroxylate genistein at the 3′ position on
the B ring to yield orobol. The metabolism of genistein
by cytochrome P450 may be another mechanism of cancer
chemoprevention. The 1A proteins are responsible for the
metabolic activation of more than 90% of the known
procarcinogenic environmental chemicals, toxins, and
toxic drugs (44). P450 1A1 is an extrahepatic enzyme
(
P.F.H.) and ES42002 (H.K.).
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