Download Dietary agents in cancer prevention: flavonoids and

Pharmacology & Therapeutics 90 (2001) 157 – 177
Associate editor: S.T. Mayne
Dietary agents in cancer prevention: flavonoids and isoflavonoids
Diane F. Birt*, Suzanne Hendrich, Weiqun Wang
Department of Food Science and Human Nutrition, 2312 Food Sciences Building, Iowa State University, Ames, IA 50011, USA
Abstract
Flavones and isoflavones may play a prominent role in cancer prevention since these compounds are found in numerous plants that are
associated with reduced cancer rates. This article reviews recent epidemiological and animal data on isoflavones and flavones and their role in
cancer prevention. It covers aspects of the bioavailability of these dietary constituents and explores their mechanism of action. Human
epidemiology data comes primarily from studies in which foods rich in isoflavones or flavones are associated with cancer rates. This
approach has been particularly useful with isoflavones because of their abundance in specific foods, including soy foods. The bioavailability
of flavones and isoflavones has been shown to be influenced by their chemical form in foods (generally glycoside conjugates), their
hydrophobicity, susceptibility to degradation, the microbial flora of the consumer, and the food matrix. Some information is available on how
these factors influence isoflavone bioavailability, but the information on flavones is more limited. Many mechanisms of action have been
identified for isoflavone/flavone prevention of cancer, including estrogenic/antiestrogenic activity, antiproliferation, induction of cell-cycle
arrest and apoptosis, prevention of oxidation, induction of detoxification enzymes, regulation of the host immune system, and changes in
cellular signaling. It is expected that some combination of these mechanisms will be found to be responsible for cancer prevention by these
compounds. Compelling data suggest that flavones and isoflavones contribute to cancer prevention; however, further investigations will be
required to clarify the nature of the impact and interactions between these bioactive constituents and other dietary components. D 2001
Elsevier Science Inc. All rights reserved.
Keywords: Flavones; Isoflavones; Cancer; Epidemiology; Bioavailability; Mechanisms
Abbreviations: ACF, aberrant crypt foci; Apc, adenomatous polyposis coli; AOM, azoxymethane; DMBA, dimethylbenz(a)anthracene; GI, gastrointestinal;
GST, glutathione-S-transferase; LDL, low-density lipoprotein; MNU, methylnitrosourea; ODMA, O-desmethylangolensin; QR, NAD(P)H:quinone reductase
(EC 1.6.99.2); TPA, 12-0-tetradecanoyl-phorbol-13-acetate.
Contents
1.
2.
3.
4.
5.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Epidemiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Anticarcinogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Bioavailability and metabolism of dietary flavonoids and isoflavonoids . . . . . . . . . .
4.1. Application of general principles of bioavailability to flavonoids and isoflavonoids
4.2. Apparent absorption and disposition kinetics of flavonoids and isoflavonoids . . .
4.3. Mammalian biotransformation of isoflavones and flavonoids . . . . . . . . . . . .
4.4. Gut microbial biotransformation of isoflavones and flavonoids . . . . . . . . . . .
4.5. Influence of other dietary components on isoflavone bioavailability . . . . . . . .
Potential mechanisms for flavonoid and isoflavonoid inhibition of cancer . . . . . . . . .
5.1. Estrogenic and antiestrogenic activity . . . . . . . . . . . . . . . . . . . . . . . .
5.2. Antiproliferation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3. Cell cycle arrest and apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.4. Antioxidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.5. Induction of detoxification enzymes . . . . . . . . . . . . . . . . . . . . . . . . .
* Corresponding author. Tel.: 515-294-3011; fax: 515-294-8181.
E-mail address: [email protected] (D.F. Birt).
0163-7258/01/$ – see front matter D 2001 Elsevier Science Inc. All rights reserved.
PII: S 0 1 6 3 - 7 2 5 8 ( 0 1 ) 0 0 1 3 7 - 1
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
158
159
161
164
164
165
166
166
168
168
168
169
169
170
171
158
D.F. Birt et al. / Pharmacology & Therapeutics 90 (2001) 157–177
5.6. Regulation of host immune function. . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.7. Other mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction
Epidemiological studies have consistently shown an
inverse association between consumption of vegetables and
fruits and the risk of human cancers at many sites (Block et al.,
1992; Messina et al., 1998; Steinmetz & Potter, 1991a). There
are many plausible mechanisms by which intake of vegetables and fruits may prevent carcinogenesis. Plant foods
contain a wide variety of anticancer phytochemicals with
many potential bioactivities that may reduce cancer susceptibility (Waladkhani & Clemens, 1998; Wattenberg, 1992a,
1992b; Steinmetz & Potter, 1991b; Adlercreutz, 1990). Flavonoids and isoflavonoids are especially promising candi-
Table 1
Chemical structures of certain commonly occurring plant flavonoid aglycones
171
172
172
dates for cancer prevention (Bravo, 1998; Kuo, 1997; Potter
& Steinmetz, 1996; Knight & Eden, 1996; Hollman et al.,
1997; Knekt et al., 1997; Adlercreutz, 1995).
Flavonoids are plant secondary metabolites, present in all
terrestrial vascular plants. Flavonoids are defined chemically
as substances composed of a common phenylchromanone
structure (C6)C3)C6), with one or more hydroxyl substituents, including derivatives (Table 1). In marked contrast to
flavonoids, isoflavonoids possess a 3-phenylchroman skeleton that is biogenetically derived from the 2-phenylchroman
skeleton of the flavonoids. Isoflavonoids are found in plants
of the subfamily Papilionoideae of the Leguminosae, which
includes soybeans (Harborne, 1989). Flavonoids and isofla-
D.F. Birt et al. / Pharmacology & Therapeutics 90 (2001) 157–177
vonoids occur commonly as ester, ether, or glycoside derivatives or mixtures thereof, and embrace over 4000 compounds (Harborne, 1989). In mammals, flavonoids and
isoflavonoids occur only through dietary intake. The average
daily human intake of flavonoids in the United Kingdom and
the United States has been estimated to be 20 mg to 1 g. These
compounds are present in fruits, vegetables, grains, nuts, tea,
and wine (Pierpoint, 1986).
Flavonoids and isoflavonoids have shown many biological properties that may account for cancer chemoprevention (Bravo, 1998; Kuo, 1996; Birt et al., 1999; Messina
et al., 1998; Adlercreutz, 1995). In recent years, considerable attention has been paid to their abilities to inhibit the
cell cycle, cell proliferation, and oxidative stress, and to
induce detoxification enzymes, apoptosis, and the immune
system. In view of heightened interest in the biological
effects of flavonoids and isoflavonoids, the time was appropriate to review the current knowledge of the epidemiology,
anticarcinogenic activity, bioavailability, and potential
mechanisms of action of flavonoids and isoflavonoids.
Building upon this foundation will facilitate development
of new strategies and approaches for cancer control.
2. Epidemiology
Studies relating flavonoid and isoflavonoid intake to
cancer rates have assessed the relationship of food groups
rich in these compounds to cancer risk. Currently, no
intervention trials have been conducted. A large body of
data has demonstrated the importance of plant intake in
reducing cancer risk (Steinmetz et al., 1994). Because
flavonoids and isoflavonoids are found in particular foods,
studies relating specific foods to cancer have been used to
develop hypotheses on the importance of flavonoids and
isoflavonoids in cancer prevention.
Flavonoids are widely distributed in plants, but little
quantitative information is available on their presence in
foods, and, thus, only a few studies have attempted to assess
the relationship between consumption of foods rich in
flavonoids and the prevention of cancer. The expanding
data on the impact of flavonoids in the prevention of animal
carcinogenesis and the impact of flavonoids on a plethora of
endpoints that have been associated with cancer prevention
have led investigators to attempt to associate the intake of
these compounds with human disease patterns. A study of
this type is being conducted in Zutphen, The Netherlands. In
this study, a cohort of 878 men were followed beginning in
1960 for 25 years, and a report was published by Hertog
et al. in 1994. This study associated the intakes of 5
flavonoids — quercetin, kampherol, myricetin, apigenin,
and luteolin — with the incidence or mortality from all
causes of cancer or with the mortality from alimentary or
respiratory tract cancers. In this study, flavonoids were
found to be primarily from tea, apples, and onions (Hertog
et al., 1992, 1993). Flavonoid intake was not associated with
159
all site cancer outcomes or with the rates of cancer in the
alimentary or respiratory tract (Hertog et al., 1994). However, the intake of flavonoids from vegetables and fruits was
only inversely associated with the risk of alimentary and
respiratory cancers, suggesting that constituents of fruits and
vegetables other than flavonoids were probably important in
the lower rates of cancer associated with these foods. In the
7 countries that were included in the Zutphen study, a 25%
decrease in coronary heart disease mortality could be
explained by flavonoid intake, but cancer risk was not
associated with flavonoid intake (Hertog et al., 1995).
A much larger and more recent study from Finland that
followed a cohort from 1967 –1991 further investigated the
association between flavonoid intake and human cancer
(Knekt et al., 1997). This study provides stronger evidence
for a protective role of flavonoids against cancer. The
incidence of cancer at all sites was inversely associated
with flavonoid intake, and this association was primarily
due to the lower rates of lung cancer in the groups with the
highest flavonoid intake (relative risk of 0.54 with a 95%
confidence interval of 0.34– 0.87, in comparison with the
lowest quartile). Fig. 1 shows the relative risk of cancer of
the lung, stomach, and colorectum in the three panels by
flavonoid quartile. The protection was greatest in individuals who were under 50 years of age (relative risk of 0.33
with a 95% confidence interval of 0.15– 0.77) and in nonsmokers (relative risk of 0.13 with a 95% confidence
interval of 0.03– 0.58) (Knekt et al., 1997). Furthermore,
this association was found not to be due to other nutrients
that have been studied for their ability to prevent cancer,
such as vitamin E, vitamin C, or ß-carotene.
Another recent study used the same database on the
flavonoid composition of foods (Hertog et al., 1992, 1993)
to assess dietary intakes of 4 flavonoids (quercetin, kampherol, myricetin, and luteolin), and specific carotenoids (acarotene, ß-carotene, lutein, and lycopene) in relation to
lung cancer in Spanish women (Garcia-Closas et al., 1998).
They observed weak associations between the greatest
lycopene or kampherol intakes and the least lung cancer
in women, but the P values did not reach statistical
significance (0.15 and 0.10, respectively).
This line of research can be criticized for the limited
number of flavonoids assessed in the fruits and vegetables,
inaccuracies in the analytical methods employed, or the
small size of the investigations (generally a few hundred
cancer cases). However, these early investigations suggest
the need for additional associative human studies. We need
to know more about the constituents of fruits and vegetables, the variability in these constituents, and the factors that
influence the constituents in order to improve interpretation
of studies of this type. The reported relationship between
flavonoid intake and cancer may be caused by some other
constituent in fruits and vegetables. We must remember the
lesson learned when, based solely on associative evidence, it
was assumed that the protective constituent in fruits and
vegetables was ß-carotene. Intervention trials have demon-
160
D.F. Birt et al. / Pharmacology & Therapeutics 90 (2001) 157–177
Fig. 1. Relative cancer risk among quartiles of flavonoid intake (Finnish
Mobile Clinic Health Survey, 1967 – 1991). Relative risk has been adjusted
for sex, age, geographic area, occupation, body mass index, smoking, and
caloric intake, vitamin C, vitamin E, ß-carotene, fiber, saturated fatty
acids, monounsaturated fatty acids, polyunsaturated fatty acids, and
cholesterol. Quartiles of flavonoid intake are: 1 ( < 2.1), 2 (2.1 – 3.2), 3
(3.3 – 4.8), and 4 ( > 4.8 mg per day in men) and 1 ( < 2.4), 2 (2.4 – 3.6), 3
(3.7 – 5.5), and 5 (>5.5 mg per day in women). Data are presented as the
relative risk ± the 95% confidence interval, and were graphed from data
from Knekt et al. (1997).
strated the fallacy of this oversimplified hypothesis (Heinonen & Albanes, 1994; Omenn et al., 1996).
Isoflavonoids are much more narrowly distributed in
foods, with soybeans being the primary human food that is
rich in these compounds. Because of this, the data associating soybean consumption with reduced cancer rates has
been used as evidence for a role of isoflavones in cancer
prevention. However, it is critical to keep in mind that
soybeans are also a rich source of trypsin inhibitor (Kennedy, 1995), other proteins with health benefits, phosphatidyl inositol, saponins, and sphingolipids, all of which
have potential health benefits. All of these soybean constituents demonstrate tumor preventative properties in animal models (Fournier et al., 1998; Birt, 2001). We recently
demonstrated that 20% by weight of dietary soy protein
significantly reduced rat intestinal mucosa levels of polyamine, a biomarker of cellular proliferation for colorectal
cancer risk. However, 0.1% of soy isoflavones (genistein
and daidzein at a 1:1 mixture) did not affect polyamine
levels (Wang & Higuchi, 2000). Recent reviews have
emphasized the complexity of soy foods and the difficulty
in assuming that associations that suggest protective properties of soy foods are due to single constituents (Fournier
et al., 1998; Birt, 2001).
Because of the association between diets in Japan and
China and lower rates of cancers, such as those of the
breast, prostate, and colon, than in Europe and the United
States, many investigators have assumed that this is due to
soy food consumption in Japan and China. Other factors in
the Asian diet may be responsible. For example, a case
control study of gastric cancer in China (Hu et al., 1988)
found that Chinese cabbage seemed to play an important
role in protecting against this common cancer. Consumption of fermented and salted soy paste was positively
associated with gastric cancer (Hu et al., 1988). Further,
a case control study of lung cancer in Chinese women in
Hong Kong indicated that fresh fruit and fish consumption
was associated with lower cancer rates (Koo, 1988). When
considering adenocarcinoma or large cell lung tumors, they
observed lesser rates of disease in people with greater
consumption of leafy green vegetables, carrots, tofu, fresh
fruit, and fresh fish. Finally, Severson et al. (1989) prospectively studied prostate cancer in Hawaiian men of
Japanese descent. Prostate cancer rates were least among
men who consumed diets abundant in rice ( P = 0.017) and
tofu (modestly protective, P = 0.054) and low in seaweed
( P = 0.017). These studies in Asian populations suggest
that soy foods, the predominant source of isoflavones, are
often associated with reductions in cancer rate, but they do
not consistently appear to be the primary protective component of the Asian diet.
In reviewing the evidence for dietary soybeans as a
protective factor against breast cancer, Wu et al. (1998)
noted the difficulties in assessing the relationship between
the level of intake and protection. Trying to combine studies
in Asia with studies from the West is particularly difficult,
since soy food consumption in Asia is much greater than in
the West. Furthermore, case control and prospective epidemiological investigations that have provided a suggestion of
protection against cancer by soy foods have not provided
adequate information on the bioactive constituents in the
soy foods, the portion size, or the other components that
may be protective in the diets of people who eat soy foods.
These investigators summarize the data on soy intake and
breast cancer as suggesting, but not consistently, that soy
foods provide some protection against breast cancer.
Because of these difficulties in relating reported intake
of soy-based foods with cancer rates, many investigations
have attempted to use biomarkers of soybean intake as
surrogate markers of soy food intake. Considering that the
purpose of this review is to consider the role of isoflavones
in cancer prevention, it is indeed fortuitous that the biomarkers used are usually isoflavones. Unfortunately, the
use of isoflavones as a marker for soy food intake leads
D.F. Birt et al. / Pharmacology & Therapeutics 90 (2001) 157–177
many casual readers to assume that isoflavones are the sole
active constituents in soy foods. Isoflavones may be
markers for another constituent or for a combination of
constituents that are the agents responsible for the cancer
prevention. Keeping this in mind, it is intriguing that some
recent investigations provide support for isoflavones as
protective against cancer. For example, Zheng et al.
(1999) assayed urinary isoflavonoids in a case control
study of breast cancer in Shanghai. Interestingly, urinary
excretion of total phenols and all individual isoflavonoids,
particularly glycetin, was lower in breast cancer patients
than in controls. The adjusted odds ratio for breast cancer
was 0.14 (relative risk of 0.02– 0.88 with a 95% confidence
interval) for women with the highest urinary excretion of
phenol and total isoflavonoids in comparison with women
in the lowest tertile.
3. Anticarcinogenesis
Studies of cancer prevention in experimental animals
have assessed the impact of a wide variety of flavonoids
and a select few isoflavones for their efficacy in inhibiting
cancer in a number of animal models.
As in other studies of dietary prevention of cancer,
models of breast and colon cancers have been prominent
in assessing cancer prevention by flavonoids and isoflavonoids. Citrus flavonoids were the focus of studies by So et
al. (1996). They determined the impact of hesperetin and
naringenin from oranges and grapefruit, respectively, and
baicalein, galangin, genistein, and quercetin from non-citrus
sources on MDA-MB-435, a human breast carcinoma cell
line. They assessed the inhibition of cell proliferation in
culture, and all of the flavonoids showed low cytotoxicity
(>500 mg/mL for 50% cell death). The IC50 (concentration
that inhibited cell proliferation by 50%) was lowest for
baicalein (5.9 mg/mL), intermediate for quercetin, hesperetin, naringenin, and galangin (10.4 – 56.1 mg/mL), and
highest for genistein (140 mg/mL). Combinations of flavonoids (1:1) generally were effective in inhibiting cell proliferation at lower doses of total flavonoid (IC50, 4.7 –9.2
mg/mL). Studies with orange juice, grapefruit juice, naringenin, and naringin, the glycosylated form of naringenin,
assessed the inhibition of mammary tumor development in
7,12-dimethylbenz(a)anthracene (DMBA)-treated female
Sprague – Dawley rats. In two experiments, the greatest
inhibition of cancer generally was observed with orange
juice (double-strength reconstitution in place of drinking
water) or naringin (0.24- to 100-g diet)-supplemented rats,
but there was considerable variability between experiments
(So et al., 1996). Further studies by this laboratory demonstrated that these compounds in media were effective against
both estrogen receptor-positive and estrogen receptor-negative breast cancer cells (IC50, 1 – 18 mg/mL) and that
double-strength orange juice was more effective than double-strength grapefruit juice in preventing the development
161
of DMBA-induced mammary cancers in rats (Guthrie &
Carroll, 1998).
The soybean isoflavones genistein and daidzein have
been studied extensively for anti-breast cancer activity
because of their estrogen receptor antagonist and agonist
activities. Studies by Constantinou et al. (1996) assessed the
ability of genistein and daidzein injections (0.8 mg daily for
180 days) against N-methyl-N-nitrosourea-induced mammary tumors in Sprague –Dawley rats. Genistein and daidzein moderately reduced the number of tumors, but only
marginally reduced the tumor incidence. They assessed
topoisomerase activity and protein tyrosine kinase activity
in normal and tumorous glands, and found that while these
activities were elevated in tumors, isoflavonoid treatment
did not alter this elevation. Thus, the inhibition of cancer by
isoflavones was independent of influences on topoisomerase or protein tyrosine kinase (Constantinou et al., 1996).
Further research by this group with cultured human breast
cancer cells demonstrated the inhibition of growth of estrogen receptor positive (MCF-7) or estrogen-receptor negative (MDA-MB-468) cells by 30 –150 mmol/L genistein and
that this inhibition was paralleled by increased expression
of maturation markers (Constantinou et al., 1998). In
addition, treatment of these cells with genistein for 6 days
with 30 mmol/L before implantation into nude mice
decreased the growth of these cells in the animal. These
investigators suggested that the inhibition of human cancer
cell growth by genistein was unrelated to the estrogenic
activity of this compound.
Another approach to assessing genistein in the prevention of breast cancer was used by Lamartiniere et al. (1995).
Considering that neonatal estrogen was known to inhibit
both spontaneous and chemically induced breast cancer, and
that diet early in life has been suspected of playing a role in
human breast cancer, these investigators treated neonatal
rats with 5-mg genistein on days 2, 4, and 6 postpartum.
They then induced mammary tumors with DMBA on day
50, and observed a delay in the development of tumors and
a reduction in the number of tumors in the rats that were
pretreated with genistein (Lamartiniere et al., 1995). Parallel
studies determined that pre-pubertal administration of 500
mg/g body weight genistein with the DMBA protocol
similarly inhibited breast cancer development. Finally,
recent studies demonstrated that exposure of rats to genistein (0, 25, and 250 mg/kg diet) from conception to 21 days
postpartum prior to treatment with DMBA at 50 days
postpartum resulted in a dose response inhibition of mammary tumors. These investigators further reported that
mammary glands at 21 and 50 days of age had fewer
terminal end buds and fewer undifferentiated terminal
ductal structures (Fritz et al., 1998). Further research
demonstrated that neonatal administration of 5 mg/pup
genistein was adverse to normal ovarian follicular development, but that pre-pubertal genistein (500 mg/g body
weight) did not appear to be toxic (Lamartiniere et al.,
1998a, 1998b).
162
D.F. Birt et al. / Pharmacology & Therapeutics 90 (2001) 157–177
In concert with parallel estrogen or genistein in early life
protecting against breast cancer, further studies determined
that genistein (750 ppm in the diet), like estrogen, when
administered during tumor development, enhanced tumor
growth of estrogen-responsive tumors (Hsieh et al., 1998).
Genistein (10 nM to 10 mM) was found to enhance the
proliferation of MCF-7 human breast cancer cells, both in
vitro and in ovariectomized athymic mice. Genistein (1 mM)
acted as an estrogen agonist in that it induced pS2, estrogenresponsive gene expression. These results suggest that
caution should be used in considering cancer prevention
by soybean isoflavones in humans. This is a particular
concern because high-potency isoflavone preparations are
now available to consumers as dietary supplements.
Studies with the drug flavone acetic acid (125 –500 mg/
kg) demonstrated that compounds in the flavonoid class
could kill transplantable rodent colon tumors (Kal et al.,
1992). Parallel studies were conducted with rats with transplantable radiation-induced lung tumors, rats with rhabdomyosarcoma BA, and mice with colon 38 tumor. The rat
lung and rhabdomyosarcoma BA tumors did not experience
a delay in growth, but the mouse colon tumor was inhibited
by intraperitoneal injections of flavone acetic acid (125 –
150 mg/kg).
Several investigations have assessed the ability of flavonoids to inhibit azoxymethane (AOM)-induced mouse or
colon tumors. Dietary quercetin (0.5 –5%) and rutin (4%)
were found to inhibit hyperproliferation and dysplasia and
to reduce colon tumor incidence in AOM-treated mice
(Deschner et al., 1991, 1993). Interestingly, the glucoside
form, rutin, was generally less effective than quercetin in
colon cancer prevention (Deschner et al., 1991). Kawamori
et al. (1995) assessed the flavonoid liquiritin in comparison
with a number of other natural phenolic compounds, and
determined that while liquiritin treatment (0.02% in the diet)
reduced some indices of proliferation, it increased others,
and it did not inhibit aberrant crypt foci (ACF), an early,
preneoplastic lesion. Tanaka et al. (1997a) determined that
diosmin and hesperidin, both alone (1000 ppm) and in
combination (900:100 ppm, respectively), inhibited indices
of cell proliferation, ACF, and colon cancer. The combination of these agents did not increase their efficacy.
Recent research in our laboratory assessed the impact of
the ubiquitous flavonoid apigenin on cell proliferation and
the cell cycle in human colon cancer cell lines (Wang et al.,
2000). SW480 [mutant ras and p53 and truncated adenomatous polyposis coli (Apc) genes], HT-29 (mutant p53 and
truncated Apc genes), and Caco-2 (mutant p53 and truncated Apc genes) were cultured with apigenin (0– 80 mM),
and cell number and cell cycle were assessed in parallel
studies. These studies suggested that the SW480 cells, with
all of the noted genes mutated, were the most sensitive to
apigenin-induced cell cycle arrest in G2/M and to the
inhibition of cell proliferation. Further studies determined
that apigenin (0.1%) in the diet of CF-1 mice that were
pretreated with AOM inhibited ACF. However, thus far, we
have no evidence for the inhibition of colon carcinogenesis,
although we have conducted three sequential carcinogenesis
experiments (Au et al., unpublished).
Genistein, the most extensively studied soybean isoflavone, was found to inhibit AOM-induced colonic ACF at
doses of 75 and 150 mg/kg (Pereira et al., 1994). Recent
studies with soy flakes, soy flour, genistein, and Ca2 +
demonstrated that soy flakes, soy flour, and genistein
reduced ACF and that genistein (0.015%) caused the greatest reduction (Thiagarajan et al., 1998). Finally, using the
min mouse model that carries a mutant Apc gene, Sorensen
et al. (1998) demonstrated that while the nonsteroidal antiinflammatory drug Sulindac2 inhibited intestinal tumors,
mice given 2 different soy isolates, either isoflavone (475
mg/kg diet) or soy protein, did not experience a reduction in
intestinal tumors.
The complexity of the relationship between topical
flavone and 7,8-benzoflavone on polycyclic hydrocarbon
skin carcinogenesis was revealed in studies reported by
Alworth and Slaga (1985). The purpose of this research,
at least in part, was to attempt to identify enzyme induction/
inhibition profiles that would be characteristic of inhibitors
of 12-0-tetradecanoyl-phorbol-13-acetate (TPA)-promoted
polycyclic hydrocarbon carcinogenesis. The results demonstrated that 7,8-benzoflavone inhibited or enhanced tumor
development, depending on the polycyclic hydrocarbon
initiator, and that flavone applications were generally inhibitory at higher (4500 nmol) topical doses, but enhancing at
low doses (450 nmol). The flavonoids robinetin, quercetin,
and myricetin were assessed in a parallel report for their
impact on polycyclic hydrocarbon initiation (Chang et al.,
1985). These flavonoids were found not to be tumor
promoters when given alone. Quercetin and robinetin (250
nmol) had little effect on tumor initiation by benzo[a]pyrene, but they weakly inhibited initiation by a diol epoxide of
benzo[a]pyrene (Chang et al., 1985). A recent report noted
that munetone, an isoflavonoid from a tropical shrub,
inhibited ornithine decarboxylase activity and thus, would
be expected to inhibit carcinogenesis in the skin of mice
(Lee et al., 1999).
A series of 14 flavonoids were examined for their ability
to inhibit the Epstein – Barr virus early antigen activation by
TPA (Konoshima et al., 1992), and 5,7,20-trihydroxyflavone
and 5,7,20,30-tetrahydroxyflavone (85 nmol) were found to
be particularly active and also to inhibit tumor promotion in
the DMBA-initiated, TPA-promoted model of skin carcinogenesis. Recent investigations have focused on the inhibition of skin carcinogenesis in the DMBA/TPA model by the
flavonoid silymarin (Lahiri-Chatterjee et al., 1999). Exceptional inhibition of skin tumor promotion was observed
when silymarin (3.6 and 12 mg) was applied prior to each
treatment with the tumor promoter TPA (75% reduction in
incidence and 97% reduction in tumor multiplicity with 12mg applications). Further, studies by this group have demonstrated the parallel inhibition of numerous markers of
cellular proliferation by silymarin.
D.F. Birt et al. / Pharmacology & Therapeutics 90 (2001) 157–177
The Birt laboratory (Birt et al., 1986, 1997; Wei et al.,
1990) conducted a series of investigations into the inhibition
of chemically induced (DMBA/TPA-treated) and UV lightinduced skin carcinogenesis by apigenin in mice (Fig. 2
shows the UV-light study). Parallel studies in the Pelling
laboratory with cultured epidermal cells (Lepley et al.,
1996) suggested that the inhibition of skin carcinogenesis
might be due to the induction of cell cycle arrest by
apigenin, as is thoroughly discussed in Section 5.3. Interestingly, the cellular concentrations of apigenin that are
achieved in mouse skin following topical treatment with
doses of apigenin (5– 10 mmol) that inhibit carcinogenesis
are similar to cellular concentrations that are achieved in
cultured cells that exhibit cell cycle arrest (20 – 30 mM)
(Lepley et al., 1996).
Studies by Wei et al. (1998) focused on the ability of the
isoflavone genistein to inhibit skin tumorigenesis. In an antiinitiation protocol, genistein (10 mM) was applied prior to
DMBA, and both tumor incidence and multiplicity were
reduced. This was associated with blockage of DMBAinduced bulky DNA-adduct formation (Wei et al., 1998). In
two anti-promotion protocols, genistein was applied prior to
TPA application, and papilloma multiplicity was reduced,
but incidence was not altered. Genistein did not modify the
proliferation marker ornithine decarboxylase, but it did
inhibit oxidative and inflammatory events (Wei et al.,
1998). Thus, it appears that while most evidence points to
flavonoids inhibiting skin tumor promotion through the
163
inhibition of cellular proliferation, the isoflavone genistein
may act by an alternative mechanism.
The inhibition of chemically induced oral cancer by
flavonoids was assessed in the rat 4-nitroquinoline 1oxide-induced model (Makita et al., 1996; Tanaka et al.,
1997b). Dietary chalcone, 2-hydroxychalcone, and quercetin were administered in the diet (500 mg/kg), either during
or following an 8-week treatment protocol with the carcinogen, and tumor rates were reduced in all flavonoid groups
(Makita et al., 1996). In the second experiment, diosmin and
hesperidin were fed alone (1000 mg/kg each) or in combination (900 and 100 mg/kg, respectively) using the above
protocols, and all groups experienced a reduction of oral
cancers (Tanaka et al., 1997b). In both of these studies, the
inhibition of oral carcinogenesis was associated with an
inhibition of cell proliferation markers (Makita et al., 1996;
Tanaka et al., 1997b).
Flavonoid inhibition of hepatocarcinogenesis by aflatoxin B1 (Nixon et al., 1984), and phenobarbital treatment
following diethylnitrosamine initiation (Lee, K. W. et al.,
1995) was assessed. Aflatoxin carcinogenesis in trout was
inhibited by 50 and 500 ppm ß-naphthoflavone, but this
inhibition was not dependent upon the induction of the
mixed-function-oxidase system (Nixon et al., 1984). Soybean isoflavone extract [containing 920 or 1840 mmol (240
or 480 mg) total isoflavones/kg diet, with approximately
equal proportions of genistein and daidzein] was fed during
phenobarbital treatment following initiation, and the devel-
Fig. 2. Inhibition by topical apigenin of squamous cell carcinoma induced by UV light in SK-1 mouse skin. UV light was administered for 11 weeks, with a
cumulative dose of 40J/cm2. Apigenin treatment at 5 mmol ( P < 0.1) or at 10 mmol ( P < 0.01) before UV light treatment increased tumor free survival. Modified
from Birt et al. (1997).
164
D.F. Birt et al. / Pharmacology & Therapeutics 90 (2001) 157–177
opment of g-glutamyltransferase and placental glutathioneS-transferase (GST) positive foci was inhibited by both
doses of isoflavone extract fed for 3 months. However,
feeding isoflavone extract for 11 months showed that while
the isoflavones inhibited altered hepatic foci in the presence
of phenobarbital, the high dose increased the development
of preneoplastic foci promoted by isoflavones alone. This
study demonstrated the importance of dose and time of
exposure in the prevention of carcinogenesis by isoflavones,
and suggested that a narrow margin of safety may exist for
isoflavones in cancer prevention.
The inhibition of prostate cancer growth and development has been the focus of several investigations. Prostate
cancer, like breast cancer, is observed less frequently in
Asia than in Western cultures, and because of this, there
has been some interest in the ability of soybeans and
genistein to prevent the development of this disease.
Unfortunately, the absence of representative models for
prostate carcinogenesis has impeded research in this area.
Studies conducted on the ability of diets containing soy
flour to inhibit tumor growth were reported by Zhang,
J.-X. et al. (1997). Feeding 33% by weight of the diet as
soy flour resulted in an 30 – 40% reduction in the growth of
transplanted Dunning R3327 prostatic adenocarcinoma in
rats. Furthermore, studies with cultured prostate cancer cell
lines suggested that genistein (1 mg/mL and 100 ng/mL,
respectively) was cytotoxic to the rat prostate cell line
MAT-lylu and the human prostate cancer cell line PC-3.
However, genistein added to the drinking water (0.07 –
0.285 mg/kg/day) failed to inhibit the growth of MAT-lylu
cells implanted into rats (Naik et al., 1994). In studies
where prostate lesions were induced by diethylstilbestrol
treatment of male rats for 3 days following birth, 7%
soybean feeding reduced the development of severe dysplasia at 9 months, but soybean feeding did not significantly reduce prostatic dysplasia at 12 months (Makela et
al., 1995). Furthermore, the ability of soybean isoflavones
to inhibit methylnitrosourea (MNU)-induced prostate seminal vesicle adenocarcinomas in Lobund – Wistar rats was
inhibited by feeding high isoflavone (1.69 mg/g)-supplemented soy diet before initiation by MNU compared with
the same diet low in isoflavones (Pollard & Luckert, 1997).
Studies using the 3,20-dimethyl-4-aminobiphenyl and testosterone propionate model in rats indicated that feeding a
soybean isoflavone mixture containing 74% genistein and
21% daidzein, at total doses of 100 and 400 ppm, reduced
the incidence of adenocarcinoma in the prostate and seminal vesicles by 50% compared with rats fed control diet
(Onozawa et al., 1999). It is noteworthy that the relevance
of the diethylstilbestrol, MNU, and 3,20-dimethyl-4-aminobiphenyl/testosterone propionate rodent models to human
prostate cancer is not clear.
Another model of carcinogenesis that was studied with
flavonoids for potential prevention was 20-methylcholanthrene-induced mouse sarcomas (Elangovan et al., 1994).
Fibrosarcoma induction in mice was inhibited by dietary
quercetin and luteolin (1% in the diet), administered either
during initiation or promotion (Naik et al., 1994).
The anti-metastatic potential of genistein and daidzein
was assessed using the pulmonary metastasis model of B16
BL6 murine melanoma cells injected into C57BL/6 mice (Li
et al., 1999). Mice were pre-fed diets containing isoflavones
(113,225,450 or 900 mmol/kg) for 2 years before and after
intravenous injection of the melanoma cells, and the isoflavone-containing diets inhibited the number of lung metastases in a dose-dependent manner (Li et al., 1999). These
intriguing data need to be followed up with different model
systems and methods of administering the flavonoids.
4. Bioavailability and metabolism of dietary flavonoids
and isoflavonoids
4.1. Application of general principles of bioavailability to
flavonoids and isoflavonoids
Bioavailability is defined operationally and pharmacologically as the proportion of the compound administered
intravenously that appears in plasma over time (measured as
area under the curve) when the compound is administered
orally. This represents the proportion of the compound that
is absorbed from the gastrointestinal (GI) tract. Bioavailability from a nutritionist’s viewpoint is often expressed as
the proportion of an ingested dose that is excreted in urine
compared with the proportion excreted in feces over time.
However, lipid-soluble compounds would not be directly
excreted in urine, but they would appear as their more
water-soluble metabolites. Notably, for lipid-soluble compounds, some fraction would enter fat stores, so ingestion
versus excretion will not tell the full story of the biological
fate of those compounds. Either plasma or urinary contents
of a compound may reflect its absorption from the GI tract.
Many compounds are extensively metabolized, and their
total bioavailability is reflected in the amounts of the parent
compound plus all bioactive metabolites. Compounds may
undergo extensive modification within the GI tract before
initial absorption or after biliary excretion before reabsorption, further metabolism, possible degradation, and ultimate
excretion. All compounds will be excreted from the body
over time, but this time course is considerably longer for
highly lipophilic compounds that are not metabolized than
for hydrophilic compounds. The time course of excretion
depends on the compound’s metabolism to more watersoluble conjugates, which are excreted through urine or bile.
Flavonoids and isoflavonoids are not very soluble in either
water or organic solvents. Even in their best solvents
(ethanol, methanol, acetonitrile), they are only moderately
soluble. Flavonoids and isoflavonoids are present in foods
mostly as glycosidic conjugates, which are more water
soluble than the parent aglycones, and may require enzymatic cleavage of the sugar moiety by mammalian or
microbial glucosidases before absorption. The phenolic
D.F. Birt et al. / Pharmacology & Therapeutics 90 (2001) 157–177
moieties of these compounds may be conjugated by UDPglucuronosyltransferases to form glucuronides or by sulfotransferases to form sulfates. The glucuronide and sulfate
conjugates are more readily transported in the blood and
excreted in bile or urine than are the parent aglycones. The
spectrum of conjugation products may be species- and
gender-dependent. These metabolites are not necessarily
biologically inert. Much work remains in order to determine
the metabolic fates and bioavailabilities of specific flavonoids and isoflavonoids. The solubility, metabolic fate of
compounds due to endogenous and exogenous biotransformation, and interaction of the compounds with other dietary
components all determine isoflavone and flavonoid bioavailability (Hendrich et al., 1998a, 1998b).
4.2. Apparent absorption and disposition kinetics of
flavonoids and isoflavonoids
The absorption of isoflavones by the GI mucosa seems
to partly depend on the relative hydrophobicity/hydrophilicity of these compounds. There is no evidence for
facilitated or active transport of isoflavones. Isoflavone
aglycones are of appropriate molecular weight (250 g/
mol) to permit their diffusion. Isoflavone glycosides predominate in foods, but isoflavone glycosides have not been
detected in human blood plasma or urine (Xu et al., 1994,
1995; King & Bursill, 1998). Human intestinal or gut
microfloral glycosidases seemingly cleave these moieties
before the isoflavones can be absorbed. Isoflavone aglycones elute in the following order under the conditions we
commonly use for reverse-phase HPLC of these compounds: daidzein, then slightly later glycitein, and much
later genistein (Wang & Murphy, 1994; Zhang et al., 1999b;
Xu et al., 1994). Although genistein has three hydroxyl
groups and daidzein only two, a hydrogen bond is created
in genistein’s structure, increasing its hydrophobicity compared with daidzein. Consistent with its greater hydrophobicity, genistein seems to be retained in the human
body for longer times than is daidzein. Humans fed soymilk
containing 13% more genistein than daidzein (predominantly as their glycosides) had apparent peak plasma
genistein concentrations of 50% greater than peak daidzein
concentrations (at 6 hr after dose). Genistein plasma concentrations were 2-fold greater than daidzein, even at 24 hr
after dosing (Zhang et al., 1999b). However, genistein
seems to be less well-absorbed overall than is daidzein
because the relative proportion of ingested daidzein
excreted in urine is 2- to 3-fold greater than is the
proportion of ingested genistein excreted in urine in numerous studies of isoflavone disposition after soy food feeding
(Xu et al., 1994, 1995, 2000; Tew et al., 1996; Zhang et al.,
1999b). This difference in urinary disposition may be due
to greater gut microbial degradation of genistein than of
daidzein (Xu et al., 1995) after the initial biliary excretion
of the compounds (Sfakianos et al., 1997), rather than to the
compounds’ solubility differences. Daidzein and glycitein
165
are of similar apparent absorbability, as reflected in percentage of ingested dose that is excreted in urine [48% vs.
47% of ingested dose of daidzein or glycitein, respectively,
after either soymilk or soygerm (glycitein-rich) was fed to
human subjects] (Zhang et al., 1999b, erratum published in
2001). This was expected from their relatively similar
retention times during HPLC. For unknown reasons, the
relative retention of glycitein in blood plasma is similar to
that of daidzein in men and significantly less than daidzein
in women (Zhang et al., 1999b). Glycitein degradation by
gut microorganisms has not been studied. Relative absorption and disposition of isoflavones seems to be determined
by relative solubility and susceptibility to degradation by
gut microorganisms.
Flavonoids are seemingly more widely dispersed in the
food supply and are of more varied composition than are
isoflavones. For these reasons, their absorption and metabolism have been less completely characterized. Recent
reviews have covered flavonoid bioavailability (Wiseman,
1999; Hollman & Katan, 1997, 1998). Studies of quercetin
disposition in ileostomy patients showed the apparent
absorption of quercetin to be 3-fold greater (50% of ingested
dose) after ingestion of quercetin predominantly in glycosidic form from onions than that of a similar dose of
quercetin aglycone or of quercetin rutinoside (17 and 24%
of ingested doses, respectively) (Hollman et al., 1995).
While the urinary excretion of quercetin differed among
treatments to the same extent, the total quercetin excreted
was 0.3% of the ingested dose. Thus, nearly all of the
absorbed quercetin must have remained in the body over the
13 hr of measurement, suggesting that quercetin has a long
elimination half-life. However, a randomized crossover
design study showing greater human plasma concentrations
of quercetin glucoside than of quercetin rutinoside described
plasma elimination half-lives of 22 and 28 hr for these
compounds, respectively (Hollman et al., 1999). To obtain a
complete picture of quercetin bioavailability, urinary and
fecal (or ileostomy) excretion and plasma concentrations
over time need to be compared within the same study. Aziz
et al. (2000) studied human plasma and urinary contents of
the flavonols quercetin and isorhamnetin after ingestion of
onions by five subjects. Peak plasma concentrations for
quercetin and quercetin and isorhamnetin glucosides
occurred at 1.5 –2 hr after ingestion. Over 24 hr, urinary
excretion of isorhamnetin glucoside (a minor onion flavonol) was 17%, but the urinary excretion of quercetin and
quercetin glucoside, the major onion flavonols, was < 1% of
ingested dose. Thus, for these flavonols, the glucoside form
seems to be absorbed intact, but quercetin is absorbed to a
very limited extent. Thus, the general, although limited, data
at this time suggests that flavonoids may be far less
bioavailable to humans than are isoflavones. Ingestion of
flavonoids and isoflavones results in micromolar plasma
concentrations of these compounds. The biological effects
of these compounds should be studied within this concentration range for nutritional relevance. The effects of fla-
166
D.F. Birt et al. / Pharmacology & Therapeutics 90 (2001) 157–177
vonoid glucosides, but probably only isoflavone aglycones,
should be investigated.
These compounds may also be investigated as potential
pharmaceuticals. A Phase 1 clinical and pharmacokinetic
trial was conducted with the synthetic flavonoid flavone
acetic acid (Havlin et al., 1991). This agent has been
extensively studied for its therapeutic potential against solid
tumors. Although the mechanism of action of flavone acetic
acid is unknown, it has been shown to induce DNA damage
in tumors, augment natural killer (NK) immunity, and act as
an anti-angiogenesis factor (Zwi et al., 1989; Havlin et al.,
1991). Thirty-eight patients were treated weekly for 4 weeks
with doses from 0.33 to 12.5 g/m2. Hypertension was
identified as a dose-limiting effect with treatment at doses
of 10 g/m2 and above. Linear pharmacokinetic parameters
were observed, and a dose of 8 g/m2 was recommended for
Phase 2 trials (Havlin et al., 1991).
4.3. Mammalian biotransformation of isoflavones and
flavonoids
Isoflavones and flavonoids may be rapidly and predominately glucuronidated in the GI mucosa, if genistein can be
considered to be a model for all of these phenolic compounds (Zhang et al., 1999a). Further, glucuronidation
occurs in the liver. Genistein undergoes biliary excretion,
with more than 70% of a dose recovered in bile within 4 hr
after dosing in rats. Although genistein may be wellabsorbed initially, a maximum of 25% of an oral genistein
dose would be eliminated in rat urine. About 20 –25% of an
oral dose of genistein (predominantly as its glucoside from
soy foods) is recovered in human urine (Watanabe et al.,
1998; Zhang et al., 1999b). Genistein may be excreted to a
greater extent in bile than are daidzein or glycitein, based on
urinary recovery data in rats (Sfakianos et al., 1997) and
humans (Xu et al., 1994, 1995; Zhang et al., 1999b). The 70-glucuronides of daidzein and genistein follow the retention of the parent isoflavones (genistein glucuronide being
the more hydrophobic of the two) (Zhang et al., 1999a).
Greater biliary excretion of genistein than daidzein (mostly
as conjugates) would be expected because the biliary route
would be somewhat more hydrophobic than the urinary
route of excretion.
The presence of hydroxylated and methylated genistein
metabolites correlated positively with inhibition of cancer
cell proliferation, but genistein sulfates were not associated
with antiproliferative effects of genistein, suggesting that
some types of metabolism of the isoflavones may be
crucial for their action (Peterson et al., 1998). Genistein
and daidzein glucuronides were an order of magnitude less
estrogenic than their respective isoflavone aglycones, as
seen in their binding to mouse uterine cytosolic estrogen
receptor (Zhang et al., 1999a). The isoflavone glucuronides also modestly enhanced human NK cell activity in
vitro, exerting effects similar to genistein aglycone. The
glucuronides were effective and nontoxic over a wider
range of concentration than was genistein. As little as 0.1to 0.5-mM isoflavone or isoflavone glucuronide enhanced
NK cell activity, levels that are achievable in plasma after
consumption of soy foods (Xu et al., 1994, 1995; Zhang
et al., 1999a).
Phenolics are probably rapidly conjugated by UDPglucuronosyltransferases and/or sulfotransferases. Such
conjugates seem not to be biologically inert, but may
be less toxic than the aglycone forms (Zhang et al.,
1999a). It may prove important to know the proportion
of the total flavonoid or isoflavone absorbed that is
converted into specific conjugated forms. Aziz et al.
(2000) did not specifically measure isorhamnetin or quercetin conjugates. It is possible, for example, that isorhamnetin conjugates co-chromatograph with the isorhamnetin
glucoside, which might significantly change the picture of
the biologically active forms of these compounds. In vitro
studies of flavonoid and isoflavone effects might be
misleading, unless the metabolism of the parent compound and the effects of the metabolites are considered.
Characterization of physiologically relevant metabolite
levels and patterns of metabolism is likely to be necessary
to determine the mechanisms of action of flavonoids
and isoflavones.
4.4. Gut microbial biotransformation of isoflavones and
flavonoids
Isoflavone and flavonoid activity may also be altered
by microbial biotransformation. Equol, an estrogenic isoflavone, is produced from daidzein in some individuals
after a few days of repeated exposure to soy foods
(Setchell et al., 1984). One of 6 men fed soy for several
weeks was an equol producer (Lu et al., 1995), but 4 of 6
women fed soy for several weeks were equol producers
(Lu et al., 1997). Thirty-five percent of the men and
women (n = 30 per gender) excreted equol after 3 days
of soy feeding, with no significant gender difference
(Lampe et al., 1998). After feeding soy flour to 12
subjects for 3 days, equol production was inversely related
to the production of O-desmethylangolensin (ODMA),
another daidzein metabolite produced during gut fermentation (Kelly et al., 1993). Several other urinary isoflavone
metabolites were detected, and they were likely to have
been derived from gut microbial fermentation, including 6hydroxy-ODMA, dehydro-ODMA, dihydrogenistein, two
isomers of tetrahydrodaidzein, and dihydrodaidzein. The
health significance of these metabolites is unclear. However, in a breast cancer case-control study in Shanghai,
breast cancer patients had lower urinary levels of the two
major isoflavone metabolites, ODMA and equol, as well
as of the three major soy aglycones, in comparison with
matched controls (Zheng et al., 1999). Any isoflavone
metabolite present in measurable amounts might be at least
a qualitative biomarker of soy intake. Estrogenic isoflavone metabolites, e.g., equol, could be useful in clarifying
D.F. Birt et al. / Pharmacology & Therapeutics 90 (2001) 157–177
the mechanisms of soy’s action, i.e., whether estrogenicity
was especially important or not. Quantifying soy intake
and health effects by examining the isoflavones and their
metabolites is complicated by the gut microbial diversity
that seems to be at least partly responsible for major
differences in isoflavone metabolism among individuals.
Likewise, flavonoids are known to be metabolized by gut
microflora (Griffiths & Smith, 1972). Metabolism by gut
microflora is likely to affect the bioavailability and effects
of phenolics in general.
Interindividual variation in isoflavone metabolism and
degradation by gut microorganisms seems to follow certain
patterns. When 7 women were fed 3 soymilk meals over 1
day, 2 women consistently showed 10- to 20-fold greater
fecal excretion of isoflavones in feces compared with the
other 5 subjects. This paralleled 2- to 3-fold greater urinary
and plasma levels of isoflavones in the ‘‘high excretors’’
compared with the other subjects (Xu et al., 1995). In vitro
isoflavone degradation by a human fecal sample was shown
to occur rapidly for both genistein and daidzein (degradation
half-lives of 3.3 and 7.5 hr, respectively). The more rapid
degradation of genistein may account for the seemingly
lesser absorption of genistein than of daidzein (Xu et al.,
1994, 1995; Zhang et al., 1999a, 1999b). Rat intestinal
bacteria degrade flavonoids and isoflavonoids with a 5-OH
group, such as genistein, to a much greater extent than
structures lacking the 5-OH group, such as daidzein (Griffiths & Smith, 1972). This might partly account for the
reduced bioavailability of some flavonoids, as well as
genistein, in comparison with daidzein. Human gut microorganisms seem to possess similar ability, based on relative
apparent absorption of the compounds and their relative
fecal degradation (Xu et al., 1995).
Twenty men and women were sorted into three distinct
isoflavone disappearance phenotypes when examined using
in vitro assays with fresh fecal samples. For example,
disappearance rate constants for genistein were 0.023 hr 1
(high degrader, n = 5), 0.163 hr 1 (moderate degrader,
n = 10), and 0.299 hr 1 (low degrader, n = 5) (Hendrich et
al., 1998a, 1998b). These phenotypes were relatively constant when reexamined 10 months later. Disappearance rate
constants for genistein were 0.049 hr 1 (high degrader,
n = 5), 0.233 hr 1 (moderate, n = 4), and 0.400 hr 1 (low,
n = 5). Twelve of 14 subjects who remained in the study after
10 months maintained their initial isoflavone disappearance
phenotype. Eight men of varying degradation phenotypes
who were fed soymilk showed plasma isoflavone contents
that correlated negatively and significantly ( P < 0.05) with
the disappearance rate constants, r = 0.88 for daidzein and
r = 0.74 for daidzein (Wang, 1997). Part of the interindividual variability in isoflavone disposition might be
accounted for by variation in gut microbial isoflavone
disappearance rate. When subjects of moderate disappearance phenotypes were chosen for an isoflavone bioavailability study, the variation between subjects in urinary
excretion of isoflavones was about 7-fold (Zhang et al.,
167
1999b). In 14 randomly selected subjects fed different doses
of soy protein for a 9-day interval, within treatment variation
between subjects in isoflavone excretion was about 13-fold
(Karr et al., 1997). Selection for isoflavone disappearance
phenotype may lessen inter-subject variation.
Thirty-five Caucasian and 35 Asian women were
studied to characterize factors influencing isoflavone disappearance phenotype (Zheng, 2000). The Asian subjects
(mostly recent immigrant Chinese) were more likely to
express high rates of isoflavone disappearance. Although
Asian and Caucasian subjects differed significantly in body
size (Caucasian > Asian), physical activity (Caucasian >
Asian), red meat intake (Asian 3-fold greater than Caucasian), dairy intake (Caucasian > Asian), and insoluble
dietary fiber intake (Caucasian, 3 g per day, vs. Asian, 1
g per day), none of these differences affected isoflavone
disappearance phenotype. Asians of the low-rate isoflavone
disappearance phenotype showed 3-fold greater urinary
excretion (apparent absorption) of genistein after soymilk
feeding than did Asians of the high isoflavone disappearance rate phenotype and all Caucasians. Asians having low
rates of isoflavone disappearance from in vitro incubations
of fecal samples had significantly more rapid gut transit
time (40 hr) than did Asians of the high-rate isoflavone
disappearance phenotype (60 hr) or Caucasians (both
phenotypes > 80 hr). This suggests that gut transit time
might significantly affect isoflavone and flavonoid bioavailability. Perhaps certain microorganisms normally
present in some human guts affect gut transit time, just
as some pathogenic organisms can, but to a lesser extent
and with benign or even beneficial results. More rapid
transit time might prevent flavonoid-degrading organisms
from growing and/or exerting their effects to a great extent.
Isoflavone and flavonoid-degrading microorganisms in
vivo in humans remain to be identified. Some Clostridia
strains cleave the C-ring of flavonoids and isoflavones
(Winter et al., 1989). Clostridia are absent from the GI
tracts of some individuals, and may be introduced during
meat consumption (Mitsuoka, 1982). Isoflavones can be
broken down into other compounds such as monophenolics.
For example, p-ethylphenol is a nonestrogenic metabolite of
genistein that is identified in ruminants (Verdeal & Ryan,
1979). Methyl p-hydroxyphenyllactate is a flavonoid metabolite that blocks nuclear estrogen receptor binding, inhibiting the growth of MCF-7 breast cancer cells in vitro
(Markaverich et al., 1988). Further support for involvement
of intestinal microorganisms in flavonoid metabolism comes
from a study of 11 humans fed a standardized horsetail
(Equisetum arvense) extract containing quercetin and caffeic
acid derivatives (Graefe & Veit, 2000). Hippuric acid
(benzoate conjugated with glycine) and dihydroferulic acid
were the main urinary metabolites collected during 8 days of
extract dosing. This suggests significant bacterial metabolism of the extract components and the need to characterize
biological effects of various bacterial metabolites of flavonoids and isoflavones.
168
D.F. Birt et al. / Pharmacology & Therapeutics 90 (2001) 157–177
Characterization of their metabolism by gut microorganisms may provide insights into the mechanisms of action of
isoflavones and flavonoids. Gut microorganisms might be
key determinants of the bioavailability and efficacy of
these compounds.
4.5. Influence of other dietary components on isoflavone
bioavailability
Bioavailability of dietary constituents to some extent
depends upon interactions with other dietary components.
For example, isoflavones are associated with soy protein.
The food matrix for isoflavones does not seem to affect
isoflavone bioavailability, although more study is warranted.
Isoflavone disposition from several soy foods was compared
when single meals of tofu, tempeh, cooked soybeans, or
texturized vegetable protein were fed to women in a randomized crossover design (Xu et al., 2000). Urinary recoveries of
isoflavones over 24 hr did not differ with soy food form
(total recovery averaged 46% of ingested daidzein and 15%
of ingested genistein among all soy foods), nor did plasma
isoflavones. Women also showed similar urinary isoflavone
disposition whether they were fed tofu (high in malonyl
glycosides) or texturized vegetable protein (high in acetyl
glycosides) (Tew et al., 1996). These studies suggest that
isoflavone aglycones, glycosides, and glycosides with different side chains are similar in bioavailability. However, when
men were fed soybean pieces or tempeh for 9 days, tempeh
isoflavones (mostly aglycones) seemed to be absorbed twice
as well as were isoflavones from soybean pieces, as reflected
in urinary excretion, although the overall isoflavone bioavailability (as urinary excretion, 5 – 10%) was lower in this
study than in several other studies (urinary total isoflavone
excretion of 25– 50%) (Hutchins et al., 1995). In rats fed
equimolar doses of genistein or genistin, the plasma content
at 10– 50 hr after dosing and total urinary isoflavone excretion were similar, although genistein was more rapidly
absorbed than was genistin (King et al., 1996). It is possible
that isoflavones from fermented soy foods, such as tempeh,
natto, or miso, would be somewhat more rapidly absorbed
because the isoflavone aglycone content would be relatively
increased in comparison with unfermented soy foods. Based
on limited evidence, isoflavone glycosides and aglycones
may be similar in overall bioavailability.
Other dietary factors seem to have little influence on
isoflavone bioavailability. Choice of background diet did
not affect isoflavone bioavailability when soymilk was fed
at three specific times on a single day to women given
three different background diets (diets provided totally by
the investigators versus self-selected diets eaten at the
specific times versus ad libitum diets) in a randomized
crossover design. No significant differences were noted
among treatments with respect to plasma or urinary isoflavone content (Xu et al., 2000). In 30 men and 30 women
who were fed soy protein powder for 4 days, greater dietary
fiber and total carbohydrate intake was associated with
equol production in women, but not in men who consumed
greater total dietary fiber than did women (Lampe et al.,
1998). In women, 40 g of dietary wheat fiber consumed
during a single day significantly suppressed urinary genistein excretion by 20% after a soy milk meal compared with
15 g dietary fiber (Tew et al., 1996). Thus, wheat fiber has
only a modest effect on isoflavone bioavailability. The
effects of other dietary factors on isoflavone bioavailability
remain to be studied. Given the structural similarities
between flavonoids and isoflavones, dietary ingredient
interactions might be expected to have little influence on
flavonoid bioavailability, as shown for isoflavones. However, this remains to be seen.
5. Potential mechanisms for flavonoid and isoflavonoid
inhibition of cancer
5.1. Estrogenic and antiestrogenic activity
The estrogenic activity of isoflavonoids was first noted in
the 1940s when clover pastures rich in isoflavones were
proposed to be responsible for infertility of sheep in Western
Australia (Bennets et al., 1946). Since then, a list of
isoflavonoids, including genistein, daidzein, and formononetin, have been shown to be estrogenic agonists in various
animal models (Moule et al., 1963; Farnsworth et al., 1975).
By using an estrogen receptor-dependent transcriptional
response assay, Miksicek (1993) reported that commonly
occurring flavonoids also had estrogenic activity. Of the
flavonoids and isoflavonoids tested in this sensitive assay,
the order of estrogenicity was genistein > kampherol >
naringenin > apigenin > daidzein > biochanin A > formononetin > luteolin > fisetin > catechin/taxifolin > hesperetin
(Miksicek, 1995). Some properties of soy isoflavones, such
as preventing osteoporosis (Anderson, 1999), improving
menopausal symptoms (Brzezinski et al., 1997), and lowering cholesterol levels (Lichtenstein, 1998), may suggest an
estrogen-related mechanism.
The estrogenic potency of all the reported isoflavonoids
and/or flavonoids is extremely weak, 103- to 105-fold less
than 17-ß-estradiol (Davis et al., 1998). Since the circulating
concentrations of isoflavone aglycones may be 100-fold
greater than estradiol, isoflavones and flavonoids may be
antiestrogenic, depending on the level of natural estrogens.
Isoflavonoids and/or flavonoids might counteract endogenous estrogens through competitive binding to estrogen
receptors. The relative binding affinity of these compounds
for the estrogen receptor (as their aglycone forms) is 0.05–
1% of the binding affinity of 17-ß-estradiol (Shutt & Cox,
1972). We examined the effect of formononetin on mammary glandular tissue and found that the estrogenic potency
of formononetin appears extremely weak and that it is
proportional to its estrogen receptor-binding capacity (Wang
et al., 1995). On the basis of both weak estrogenic activity
and low estrogen receptor-binding capacity, isoflavonoids
D.F. Birt et al. / Pharmacology & Therapeutics 90 (2001) 157–177
and/or flavonoids might be unlikely to exert significant
cancer prevention via antiestrogenic mechanism(s). However, a recent study of isoflavone estrogenicity in vivo
(mouse uterine growth assay) and in vitro (mouse uterine
cytosolic estrogen receptor binding) showed that glycitein
was about 3-fold more potent than genistein in vivo, but
about 20-fold less potent than genistein in vitro (unpublished). This suggests that bioavailability in vivo and in
vitro may differ significantly. The efficacy of isoflavones
and flavonoids as estrogen agonists or antagonists in vivo
requires much more study.
169
isoflavone analogues, at 0.1 – 25 mg/mL, inhibited intestinal
epithelial cell proliferation and induced apoptosis in vitro.
An inhibition of human breast cancer cell proliferation and
delay of mammary tumorigenesis by flavonoids was
reported by So et al. (1996).
Although the antiproliferative effects of flavonoids and
isoflavonoids in cultured cells appear well established,
relatively little data have been published regarding the
antiproliferative activity in vivo, and virtually nothing is
known regarding the clinical relevance of this bioactivity.
5.3. Cell cycle arrest and apoptosis
5.2. Antiproliferation
Dysregulated proliferation appears to be a hallmark of
increased susceptibility to neoplasia. Cancer prevention is
generally associated with inhibition, reversion, or retardation of cellular hyperproliferation. It is well known that
dietary flavonoids and isoflavonoids behave as general cell
growth inhibitors. One of their biological properties in
plants, in fact, is providing resistance to fungal or bacterial
growth as phytoalexins (Dewick, 1988; Bohm, 1980).
Although most flavonoids and isoflavonoids appear nontoxic to humans and animals, they have been demonstrated
to inhibit proliferation in many kinds of cultured human
cancer cell lines. Kandaswami et al. (1991), for example,
reported antiproliferative effects of four citrus flavonoids
(quercetin, taxifolin, nobiletin, and tangeretin, at 2– 8 mg/
mL for 3– 7 days) on squamous cell carcinoma HTB43.
Piantelli et al. (1993) demonstrated antiproliferative activity
of 10 mM quercetin on meningioma cells. Kuo (1996)
showed antiproliferative potency of 5 flavonoids and 2
isoflavonoids (0– 100 mM) on colon carcinoma HT29 and
Caco-2 cell lines with apoptosis induction mechanisms.
Hirano et al. (1994) found inhibitory effects of 8 of the 28
tested flavonoids on leukemia HL-60 cells (IC50 < 100 ng/
mL) via a nontoxic mechanism. Le Bail et al. (1998)
suggested antiproliferative activity of certain flavonoids
and genistein at high concentrations (50 mM) on breast
cancer MCF-7 through estrogen receptor-independent
mechanisms. Kawaii et al. (1999) investigated antiproliferative activities of 27 citrus flavonoids, at 40 mM, on several
tumor cell lines, including lung carcinoma A549 and
gastric TGBC11TKB cancer cells, and found that they
inhibited proliferation of cancer cell lines, but that they
did not significantly affect proliferation of human normal
cell lines. Fotsis et al. (1997) studied the inhibition of cell
proliferation and angiogenesis by flavonoids in 6 different
cancer cell lines, and noted that the IC50 of active flavonoids was in the low micromolar range, physiologically
available concentrations. The influence of flavonoids on
lymphocyte proliferation was studied by Lee, S. J. et al.
(1995). They observed a nonreversible inhibition of concanavalin A-induced lymphocyte proliferation by quercetin
and apigenin, with IC50s of 10 mM and 3 mM, respectively.
Booth et al. (1999) reported that genistein and synthetic
The observed antiproliferative properties of flavonoids
and isoflavonoids suggests that these compounds may
inhibit the cell cycle or induce apoptosis. Indeed, checkpoints at both G1/S and G2/M of the cell cycle in cultured
cancer cell lines have been found to be perturbed by
flavonoids and isoflavonoids. Traganos and co-workers
(1992) demonstrated that genistein, at 5 – 20 mg/mL, produced cell cycle arrest at both the G1/S and G2/M phases in
the human myelogenous leukemia HL-60 line and the
lymphocytic leukemia MOLT-4 cell line. A later study by
Matsukawa et al. (1993) reported that genistein, up to 60
mM, arrested human gastric cancer cells at G2/M. Studies
conducted in a non-small-cell lung cancer cell line demonstrated that genistein, at 30 mM, induced G2/M arrest
through p21 up-regulation and apoptosis induction (Lian
et al., 1998). Zhou et al. (1998) demonstrated that isoflavones (genistein, genistin, daidzein, and biochanin A, at 0–
50 mM) inhibited growth of murine and human bladder
cancer cell lines by inducing cell cycle arrest, apoptosis, and
angiogenesis (Zhou et al., 1998). Other studies regarding
the induction of apoptosis by genistein at similar concentrations were reported in human prostatic cancer cell lines
(Kyle et al., 1997) and in Jurkat T-leukemia cell line
(Spinozzi et al., 1994).
Quercetin (30 –100 mM), a widely distributed flavonoid,
was reported to block the cell cycle at G1/S in human
colonic COLO320 DM cells (Hosokawa et al., 1990) and
leukemic T-cells (Yoshida et al., 1992), and to trigger
apoptosis (Wei et al., 1994b). Wei and co-workers
(1994a) reported the induction of apoptosis by quercetin,
at 50 mM or higher, in several tumor cell lines, resulting in
nuclear fragmentation, condensation of nuclear chromatin,
and a subdiploid peak by DNA flow cytometry. Previous
studies conducted in our laboratory demonstrated that
apigenin (0 –80 mM), another widely distributed flavonoid,
significantly induced a reversible G2/M arrest in keratinocytes (Lepley et al., 1996), fibroblasts (Lepley et al., 1996;
Lepley & Pelling, 1997), and colonic carcinoma cell lines
(Wang et al., 2000). Studies on silymarin (0 –75 mM) found
that perturbations in cell cycle progression may account for
the anticarcinogenic effects on human prostate carcinoma
DU145 cells (Zi et al., 1998) and epidermoid carcinoma
A431 cells (Giles & Wei, 1997). Cell cycle arrest and
170
D.F. Birt et al. / Pharmacology & Therapeutics 90 (2001) 157–177
induction of apoptosis could be functionally related to
activation of p53 (Plaumann et al., 1996) and/or inhibition
of cell cycle kinase activity (End et al., 1987; De Azevedo
et al., 1996; Kyle et al., 1997). Recently, we compared the
effects of apigenin (0– 80 mM) on cell growth and cell
cycle arrest in three human colonic cancer cell lines,
SW480, Caco-2, and HT-29 (Wang et al., 2000). The
effects on both cell cycle arrest and cell growth inhibition
were strongest in SW480 cells, moderate in HT-29 cells,
and weakest in Caco-2 cells (Fig. 3). The differential
effectiveness of cell growth inhibition and cell cycle arrest
in response to apigenin in the three cell lines tested might
be related to their status of functional p53 and/or ras
genes. The results suggest that cells with mutations in
genes critical to colon cancer development may be more
sensitive to apigenin. This implies that flavonoids may be
more effective in controlling growth of tumors with certain
mutational spectra.
5.4. Antioxidation
Flavonoids and isoflavonoids might protect against cancer
and/or heart disease through inhibition of oxidative damage
(Omenn, 1995). Oxidation of DNA is likely to be an
important cause of mutations that potentially can be reduced
by dietary antioxidants. Chemically, flavonoids and isoflavonoids are one-electron donors. They serve as derivatives of
conjugated ring structures and hydroxyl groups that have the
potential to function as antioxidants in in vitro cell culture or
cell free systems by scavenging superoxide anion (Robak &
Gryglewski, 1988), singlet oxygen (Husain et al., 1987), lipid
peroxy-radicals (Torel et al., 1986), and/or stabilizing free
radicals involved in oxidative processes through hydrogenation or complexing with oxidizing species (Lewis, 1993;
Shahidi et al., 1992). Flavonoids themselves become free
radicals in the process, but their conjugated structure allows
the remaining orbital electron to be relatively inactive (Hudson & Lewis, 1983). The antioxidant activity and the structure – activity relationships of flavonoids have been
investigated intensively in many in vitro systems (Lien
et al., 1999; Yamanaka et al., 1997; Foti et al., 1996; Ratty
& Das, 1988). Antioxidant activities of isoflavonoids are also
reported (Arora et al., 1998; Mitchell et al., 1998). Quercetin,
luteolin, and genistein have been shown to inhibit oxidative
DNA damage induced by UV light irradiation in HL-60 cells
with IC50 < 1 mM (Giles & Wei, 1997; Cai et al., 1997). The
antioxidant activity of flavonoids was suggested to be related
to the number and position of hydroxyl groups (Noroozi
Fig. 3. Effect of apigenin on the cell cycle in three human colonic carcinoma cell lines. SW480, HT-29, or Caco-2 cells were respectively co-cultured with 80
mM of apigenin in the Dulbecco’s modified eagle media supplemented with 10% fetal bovine serum at 37C in a 5% CO2 atmosphere. At the times indicated on
the right of the panel, histograms of cellular DNA content were obtained by flow cytometry.
D.F. Birt et al. / Pharmacology & Therapeutics 90 (2001) 157–177
et al., 1998). However, relatively few data have been published regarding their in vivo roles.
Theoretical underpinnings for the efficacy of flavonoids
as antioxidants in vivo come from the inhibition of lowdensity lipoprotein (LDL) oxidation. Elevated LDL oxidation has been associated with coronary artery disease. An
inverse association of the dietary intake of phenolic flavonoids from red wine, with the risk of developing
cardiovascular diseases, has been observed in French
populations (Frankel et al., 1993; Renaud & De Lorgeril,
1992). We recently examined the antioxidant efficiency of
26 common dietary flavonoids, isoflavonoids, and phenolic
acids (20 – 200 mM) in a human ex vivo LDL-oxidation
model (Wang & Goodman, 1999). The antioxidative efficiency of dietary flavonoids and isoflavonoids appears to
be associated not only with their reductive capacity, but
also with their protein-binding properties. It is suggested
that the availability of flavonoids at the oxidative site on
LDL may block oxidative attack and prevent LDL oxidation in vivo. Potential protein binding and interference with
oxidative processes by flavonoids and isoflavonoids may
explain their ultimate antioxidant roles in vivo (Wang &
Goodman, 1999).
171
seaud, 1979). While some flavonoids, such as chrysin,
apigenin, luteolin, kampherol, quercetin, myricetin, and
naringenin, have high potencies and selectivities for inhibition of CYP1A isoforms (IC50 < 4 mM) (Kanazawa et al.,
1998), induction of either QR or GST activity has been
noted for certain dietary antioxidant flavonoids, such as
quercetin (50 – 100 mM in cultured cells or 1% in murine
diet) and epicatechin (25 – 100 mM) (Wang & Higuchi,
1995; Gordon et al., 1991; Benson et al., 1980; Rodgers
& Grant, 1998; Nijhoff et al., 1993).
We measured the effects of 4 prominent isoflavones upon
QR induction in a human colonic Colo205 cell line, and
found a dose-dependent induction in enzyme activity up to
6- to 8-fold after addition of genistein, at 0.01 –10.0 mM,
and up to 2- to 3-fold induction with biochanin A, at 1.0–
10.0 mM. The observed induction of QR by isoflavones
appears through the promotion of QR mRNA expression
and stabilization of mRNA levels (Wang et al., 1998).
Current scientific opinion suggests that the optimal conditions for preventing carcinogen activation would couple
inhibition of Phase I metabolism with activation of Phase
II metabolism, and we are presently not aware of flavonoids
or isoflavones that have been shown to have this favorable
induction/inhibition profile.
5.5. Induction of detoxification enzymes
5.6. Regulation of host immune function
Another proposed mechanism for protection against
cancer by dietary flavonoids and isoflavonoids may include
induction of Phase II detoxification enzymes in cells.
Modification of cellular detoxification enzymes could be a
major mechanism for protecting against the toxic and neoplastic effects of carcinogens (Talalay, 1989; Wattenberg,
1992b; Talalay et al., 1995). Many environmental carcinogens require metabolism to their fully carcinogenic forms.
They are often metabolized to proximate carcinogens by
Phase I enzymes, e.g., cytochromes P450, which catalyze
oxidative reactions (Talalay, 1989). The oxidized metabolites of potentially carcinogenic xenobiotics are then detoxified by Phase II metabolizing enzymes into the forms that
are relatively inert and more easily excreted (Talalay et al.,
1995). There is considerable evidence that induction of
Phase II detoxification enzymes can modulate the threshold
for chemical carcinogenesis and then increase cellular
resistance to carcinogen exposure (Talalay, 1989; Wattenberg, 1992b; Talalay et al., 1995). Both NAD(P)H:quinone
reductase (EC 1.6.99.2) (QR) and GST (EC 2.5.1.18), for
example, are Phase II detoxifying enzymes. They are widely
induced in many cells coordinately with xenobiotic exposures. QR is a major enzyme of xenobiotic metabolism that
carries out obligatory two-electron reductions and thereby
protects cells against mutagenicity and carcinogenicity
resulting from free radicals and toxic oxygen metabolites
generated by the one-electron reductions catalyzed by
cytochromes P450 and other enzymes (Ernster, 1967).
GST detoxifies a number of carcinogenic electrophiles by
catalysis of the conjugation with reduced glutathione (Chas-
The role of the host immune function has become
increasingly important in our understanding of the mechanisms that are involved in cancer prevention. The
enhancement of host immune function by dietary antioxidants may be beneficial to cancer prevention (Hughes,
1999; Appelbaum, 1992; Lowell et al., 1990; Watson,
1986). Since phagocytic cells produce reactive oxygen
species as part of the body’s defense against infection,
adequate intake of antioxidants is required to prevent
damage by oxidants to the immune cells themselves.
Middleton and Kandaswami have demonstrated that a
number of immune cell systems do not appear to be
affected significantly by flavonoids, notably quercetin,
while they are resting (Middleton, 1998). However, once
a cell becomes activated by a physiological stimulus, a
flavonoid-sensitive substance is generated and interaction
of flavonoids with that substance dramatically alters the
outcome of the activation process (Middleton, 1998; Middleton & Kandaswami, 1992). By using a murine model,
we demonstrated that the isoflavone daidzein administrated
orally at doses of 20– 40 mg/kg body weight stimulated
murine nonspecific immunity, activated humoral immunity,
and enhanced cell-mediated immunity (Zhang, R. et al.,
1997). In addition, we examined the influence of daidzein,
genistein, and combinations of these isoflavones on the
proliferation of murine splenocytes activated with mitogens and on the secretion of IL-2 and IL-3. We found that
daidzein at physiologically relevant concentrations (0.01 –
10 mM) potentiates lymphocyte activation, suggesting that
172
D.F. Birt et al. / Pharmacology & Therapeutics 90 (2001) 157–177
the immunostimulatory effects of daidzein may be
involved in cancer chemoprevention (Wang et al., 1997).
Although the clinical relevance of this finding is not clear,
we also observed that daidzein and genistein glucuronides
and genistein (in nutritionally relevant concentrations,
0.1 –10 mM) enhanced activation of NK cells in vitro
(Zhang et al., 1999a). The glucuronides were less toxic to
NK cells than was genistein, which inhibited NK activity
at 50 mM.
growth, the ability of flavonoid or isoflavonoid combinations to prevent cancer progression has not been adequately
studied. Clinical studies using these dietary agents are
scarce. Further investigations into the potential role of
flavonoids and isoflavonoids in cancer prevention and/or
therapy are warranted.
5.7. Other mechanisms
Adlercreutz, H. (1990). Western diet and Western diseases: some hormonal
and biochemical mechanisms and associations. Scand J Clin Lab Invest
50 (Suppl. 201), 3 – 23.
Adlercreutz, H. (1995). Phytoestrogens: epidemiology and a possible role
in cancer protection. Environ Health Perspect 103 (suppl. 7), 103 – 112.
Alworth, W. L., & Slaga, T. J. (1985). Effects of ellipticine, flavone, and
7,8-benzoflavone upon 7,12-dimethylbenz[a]anthracene, 7,14-dimethyl-dibenzo[a,h]anthracene and dibenzo[a,h]anthracene initiated skin tumors in mice. Carcinogenesis 6, 487 – 493.
Anderson, J. J. B. (1999). Plant-based diets and bone health: nutritional
implications. Am J Clin Nutr 70, 539S – 542S.
Appelbaum, J. W. (1992). The role of the immune system in the pathogenesis of cancer. Semin Oncol Nurs 8, 51 – 62.
Arora, A., Nair, M. G., & Strasburg, G. M. (1998). Antioxidant activities of
isoflavones and their biological metabolites in a liposomal system. Arch
Biochem Biophys 356, 133 – 141.
Aziz, A. A., Edwards, C. A., Lean, M. E. J., & Crozier, A. (2000). Absorption and excretion of conjugated flavonols, including quercetin-40O-ß-glucoside and isorhamnetin-40-O-ß-glucoside by human volunteers
after the consumption of onions. Free Radic Res 29, 257 – 269.
Bennets, H. W., Underwood, E. J., & Shier, F. L. (1946). A specific breeding problem of sheep on subterranean clover pastures in western Australia. Aust Vet J 22, 2 – 12.
Benson, A. M., Hunkeler, M. J., & Talalay, P. (1980). Increase of
NAD(P)H:quinone reductase by dietary antioxidants: possible role in
protection against carcinogenesis and toxicity. Proc Natl Acad Sci USA
77, 5216 – 5220.
Birt, D. F. (2001). Soybeans and cancer prevention: a complex food and a
complex disease. In Proceedings of the American Institute for Cancer
Research, New Insights into the Role of Phytochemicals. New York:
Plenum: Kluwer, in press.
Birt, D. F., Walker, B., Tibbels, M. G., & Bresnick, E. (1986). Anti-mutagenesis and anti-promotion by apigenin, robinetin and indole-3-carbinol.
Carcinogenesis 7, 959 – 963.
Birt, D. F., Mitchell, D., Gold, B., Pour, P., & Pinch, H. C. (1997). Inhibition of ultraviolet light induced skin carcinogenesis in SKH-1 mice by
apigenin, a plant flavonoid. Anticancer Res 17, 85 – 91.
Birt, D. F., Shull, J. D., & Yaktine, A. (1999). Chemoprevention of cancer.
In M. E. Shils, J. E. Olson, M. Shike, & A. C. Ross (Eds.), Modern
Nutrition in Health and Disease ( pp. 1263 – 1295). Baltimore: Williams and Wilkins.
Block, G., Patterson, B., & Subar, A. (1992). Fruit, vegetables, and cancer
prevention: a review of the epidemiological evidence. Nutr Cancer 18,
1 – 29.
Bohm, B. A. (1980). The minor flavonoids. In J. B. Harborne (Ed.), The
Flavonoids, Advances in Research Since 1980 ( pp. 329 – 388). London:
Chapman and Hall.
Booth, C., Hargreaves, D. F., Hadfield, J. A., McGown, A. T., & Potton, C. S.
(1999). Isoflavones inhibits intestinal epithelial cell proliferation and
induce apoptosis in vitro. Br J Cancer 80, 1550 – 1557.
Bravo, L. (1998). Polyphenols: chemistry, dietary sources, metabolism, and
nutritional significance. Nutr Rev 56, 317 – 333.
Brzezinski, A., Adlercreutz, H., Shaoul, R., Rosler, A., Shmueli, A., Tanos,
V., & Schenker, J. G. (1997). Short-term effects of phytoestrogen-rich
diet on postmenopausal women. Menopause 4, 89 – 94.
Numerous additional mechanisms have been suggested
for flavonoid and/or isoflavonoid inhibition of carcinogenesis. Certain studies have associated changes in protein
phosphorylation of cancer cell lines with growth inhibition
by flavonoids and isoflavonoids. For example, apigenin,
kampherol, and genistein, at 25 mM, reversed the transformed phenotype of v-H-ras-transformed NIH 3T3 cells
that was associated with a reduction in phosphotyrosine
content in the cells (Kuo et al., 1994). Further work from the
same group suggested that apigenin, at 12.5 mM, inhibits
mitogen-activated protein kinase (Kuo & Yang, 1995).
Gamet-Payrastre et al. (1999) recently reported that some
flavonoids such as quercetin blocked particular isoforms of
phosphoinositide 3-kinase or protein kinase C and their
downstream-dependent cellular responses (Gamet-Payrastre
et al., 1999). Furthermore, a number of flavonoids were
shown to be topoisomerase antagonists, of which myricetin,
quercetin, fisetin, and morin inhibited both Topo I and Topo
II, while others, such as kampherol and 40,6,7-trihydroxyisoflavone, inhibited either Topo I or Topo II (Constantinou
et al., 1994). The inhibition by genistein (20 –100 mM) on cmyc oncogene expression in colon cancer cell lines was
suggested to be related to the inhibition of tyrosine kinase
activity rather than to the inhibition of topoisomerase II
(Heruth et al., 1995). Genistein (30 –200 mg/mL) was also
reported to induce differentiation (Constantinou et al., 1990)
and inhibit angiogenesis (IC50 = 150 mM) (Fotsis et al.,
1993). Although dietary flavonoids and isoflavonoids are
usually nontoxic, some flavonoids such as quercetin were
reported to be potent mutagens (Friedman & Smith, 1984;
Czeczot et al., 1990), and some flavonoids and isoflavonoids, such as kampherol and genistein, at 1 mM, were
identified as estrogenic agonists (Miksicek, 1993, 1995).
The evidence for potentially harmful effects of flavonoids
and/or isoflavonoids is limited and has been generally
discussed in Section 3.
Considering the great variety of dietary flavonoids, it
appears extremely unlikely that any one substance is
responsible for all of the associations seen between plant
foods and cancer prevention. The specific mechanisms of
action of most flavonoids and isoflavonoids, with respect to
cancer prevention, are not clear yet, but appear to be varied,
complementary, and/or overlapping. Although many individual flavonoids have been shown to inhibit cancer cell
References
D.F. Birt et al. / Pharmacology & Therapeutics 90 (2001) 157–177
Cai, Q. Y., Rahn, R. O., & Zhang, R. W. (1997). Dietary flavonoids,
quercetin, luteolin and genistein, reduce oxidative DNA damage and
lipid peroxidation and quench free radicals. Cancer Lett 119, 99 – 107.
Chang, R. L., Huang, M.-T., Wood, A. W., Wong, C.-Q., Newmark, H. L.,
Yagi, H., Sayer, J. M., Jerina, D. M., & Conney, A. H. (1985). Effect of
ellagic acid and hydroxylated flavonoids on the tumorigenicity of benzo[a]pyrene and ( ± -7beta,8alpha-dihydroxy-9alpha,10alpha-epoxy7,8,9-10-tetrahydrobenzo[a]pyrene on mouse skin and in the newborn
mouse. Carcinogenesis 6, 1127 – 1133.
Chasseaud, L. F. (1979). The role of glutathione and glutathione S-transferases in the metabolism of chemical carcinogens and other electrophilic agents. Adv Cancer Res 29, 175 – 274.
Constantinou, S., Kiguchi, K., & Huberman, E. (1990). Induction of differentiation and DNA strand breakage in human HL-60 and K-562 leukemia cells by genistein. Cancer Res 50, 2618 – 2624.
Constantinou, A., Mehta, R., Runyan, C., Rao, K., Vaughan, A., & Moon, R.
(1994). Flavonoids as DNA topoisomerase antagonists and poisons:
structure – activity relationships. Surg Oncol 1, 2 – 3.
Constantinou, A. I., Mehta, R. G., & Vaughan, A. (1996). Inhibition of Nmethyl-N-nitrosourea-induced mammary tumors in rats by the soybean
isoflavones. Anticancer Res 16, 3293 – 3298.
Constantinou, A. I., Krygier, A. E., & Mehta, R. R. (1998). Genistein
induces maturation of cultured human breast cancer cells and prevents
tumor growth in nude mice. Am J Clin Nutr 68, 1426S – 1430S.
Czeczot, H., Tudek, B., Kusztelak, J., Szymczyk, T., Dobrowolska, B.G.-G.,
Malinowski, J., & Strzelecka, H. (1990). Isolation and studies of the
mutagenic activity in the Ames test of flavonoids naturally occurring in
medical herbs. Mutat Res 240, 209 – 216.
Davis, S. R., Murkies, A. L., & Wilcox, G. (1998). Phytoestrogens in
clinical practice. Integr Med 1, 27 – 34.
De Azevedo, W. F., Jr., Mueller-Dieckmann, H. J., Schulze-Gahmen, U.,
Worland, P. J., Sausville, E., & Kim, S. H. (1996). Structural basis for
specificity and potency of a flavonoid inhibitor of human CDK2, a cell
cycle kinase. Proc Natl Acad Sci USA 93, 2735 – 2740.
Deschner, E. E., Ruperto, J., Wong, G., & Newmark, H. L. (1991). Quercetin
and rutin as inhibitors of azoxymethanol-induced colonic neoplasia.
Carcinogenesis 12, 1193 – 1196.
Deschner, E. E., Ruperto, J. F., Wong, G. Y., & Newmark, H. L. (1993).
The effect of dietary quercetin and rutin on AOM-induced acute colonic
epithelial abnormalities in mice fed a high-fat diet. Nutr Cancer 20,
199 – 204.
Dewick, P. M. (1988). Isoflavonoids. In J. B. Harborne (Ed.), The Flavonoids, Advances in Research Since 1980 ( pp. 125 – 209). London:
Chapman and Hall.
Elangovan, V., Sekar, N., & Govindasamy, S. (1994). Chemopreventive
potential of dietary bioflavonoids against 20-methylcholanthrene-induced tumorigenesis. Cancer Lett 87, 107 – 113.
End, D. W., Look, R. A., Shaffer, N. L., Balles, E. A., & Persico, F. J.
(1987). Non-selective inhibition of mammalian protein kinases by flavinoids in vitro. Res Commun Chem Pathol Pharmacol 56, 75 – 86.
Ernster, L. (1967). DT diaphorase. In R. W. Estabrook, & M. E. Pullman
(Eds.), Methods in Enzymology ( pp. 309 – 317). New York: Academic
Press.
Farnsworth, N. R., Bingel, A. S., Cordell, G. A., Crane, F. A., & Fong, H.
H. (1975). Potential value of plants as sources of new antifertility agents
II. J Pharm Sci 64, 717 – 754.
Foti, M., Piattelli, M., Baratta, M. T., & Ruberto, G. (1996). Flavonoids,
coumarins, and cinnamic acids as antioxidants in a micellar system.
Structure-activity relationship. J Agric Food Chem 44, 497 – 501.
Fotsis, T., Pepper, M., Adlercreutz, H., Fleischmann, G., Hase, T., Montesano, R., & Schweigerer, L. (1993). Genistein, a dietary-derived
inhibitor of in vitro angiogenesis. Proc Natl Acad Sci USA 90,
2690 – 2694.
Fotsis, T., Pepper, M. S., Aktas, E., Breit, S., Rasku, S., Adlercreutz, H.,
Wähälä, K., Montesano, R., & Schweigerer, L. (1997). Flavonoids,
dietary-derived inhibitors of cell proliferation and in vitro angiogenesis.
Cancer Res 57, 2916 – 2921.
173
Fournier, D. B., Erdman, J. W., Jr., & Gordon, G. B. (1998). Soy, its
components, and cancer prevention: a review of the in vitro, animal,
and human data. Cancer Epidemiol Biomarkers Prev 7, 1055 – 1065.
Frankel, E. N., Kanner, J., German, J. B., Parks, E., & Kinsella, J. E.
(1993). Inhibition of oxidation of low-density lipoprotein by phenolic
substances in red wine. Lancet 341, 454 – 457.
Friedman, M., & Smith, G. A. (1984). Factors which facilitate inactivation
of quercetin mutagencity. Adv Exp Med Biol 177, 527 – 544.
Fritz, W. A., Coward, L., Wang, J., & Lamartiniere, C. A. (1998). Dietary
genistein: perinatal mammary cancer prevention, bioavailability and
toxicity testing in the rat. Carcinogenesis 19, 2151 – 2158.
Gamet-Payrastre, L., Manenti, S., Gratacap, M. P., Tulliez, J., Chap, H., &
Payrastre, B. (1999). Flavonoids and the inhibition of PKC and PI
3-kinase. Gen Pharmacol 32, 279 – 286.
Garcia-Closas, R., Agudo, A., Gonzalez, C. A., & Riboli, E. (1998). Intake
of specific carotenoids and flavonoids and the risk of lung cancer in
women in Barcelona, Spain. Nutr Cancer 32, 154 – 158.
Giles, D., & Wei, H. (1997). Effect of structurally related flavones/isoflavones on hydrogen peroxide production and oxidative DNA damage in
phorbol ester-stimulated HL-60 cells. Nutr Cancer 29, 77 – 82.
Gordon, G. B., Prochaska, H. J., & Yang, L. Y. S. (1991). Induction of
NAD(P)H:quinone reductase in human peripheral blood lymphocytes.
Carcinogenesis 12, 2393 – 2396.
Graefe, E. U., & Veit, M. (2000). Urinary metabolites of flavonoids and
hydroxycinnamic acids in humans after application of a crude extract
from equisetum arvense. Phytomedicine 6, 239 – 246.
Griffiths, L. A., & Smith, G. E. (1972). Metabolism of apigenin and related
compounds in the rat; metabolite formation in vivo and by the intestinal
microflora in vitro. Biochem J 128, 901 – 911.
Guthrie, N., & Carroll, K. K. (1998). Inhibition of mammary cancer by
citrus flavonoids. Adv Exp Med Biol 439, 227 – 236.
Harborne, J. B. (1989). General procedures and measurement of total phenolics. In P. M. Dey, & J. B. Harborne (Eds.), Methods in Plant Biochemistry, Vol. 1 ( pp. 1 – 28). London: Academic Press.
Havlin, K. A., Kuhn, J. G., Craig, J. B., Boldt, D. H., Weiss, G. R. Koeller,
J., Harman, G., Schwartz, R., Clark, G. N., & Von Hoff, D. D. (1991).
Phase I clinical and pharmacokinetic trial of flavone acetic acid. J Natl
Cancer Inst 83, 124 – 128.
Heinonen, O. P., & Albanes, D. (1994). The effect of vitamin E and beta
carotene on the incidence of lung cancer and other cancers in male
smokers. The Alpha-tocopherol, Beta Carotene Cancer Prevention
Study Group. N Engl J Med 330, 1029 – 1035.
Hendrich, S., Wang, G. J., Lin, H. K., Xu, X., Tew, B. Y., Wang, H. J., &
Murphy, P. A. (1998a). Isoflavones metabolism and bioavailability. In
A. Pappas (Ed.), Antioxidant Status, Diet, Nutrition and Health
( pp. 211 – 230). Boca Raton: CRC Press LLC.
Hendrich, S., Wang, G.-J., Xu, X., Tew, B. Y., Wang, H.-J., & Murphy, P.
A. (1998b). Human bioavailability of soy bean isoflavones: influences
of diet, dose, time and gut microflora. In T. Shibamoto (Ed.), Functional
Foods ( pp. 150 – 156). Washington, DC: ACS Books.
Hertog, M. G. L., Hollman, P. C. H., & Katan, M. B. (1992). Content of
potentially anticarcinogenic flavonoids of 28 vegetables and 9 fruits
commonly consumed in the Netherlands. J Agric Food Chem 40,
2379 – 2383.
Hertog, M. G. L., Hollman, P. C. H., & van de Putte, B. (1993). Content of
potentially anticarcinogenic flavonoids of tea infusions, wines, and fruit
juices. J Agric Food Chem 41, 1242 – 1248.
Hertog, M. G. L., Feskens, E. J. M., Hollman, P. C. H., Katan, M. B., &
Kromhout, D. (1994). Dietary flavonoids and cancer risk in the Zutphen
elderly study. Nutr Cancer 22, 175 – 184.
Hertog, M. G. L., Kromhout, D., Aravanis, C., Blackburn, H., Buzina, R.,
Fidanza, F., Giampaoli, S., Jansen, A., Menotti, A., Nedeljkovic, S.,
Pekkarinen, M., Simic, B. S., Toshima, H., Feskens, E. J. M., Hollman,
P. C. H., & Katan, M. B. (1995). Flavonoid intake and long-term risk of
coronary heart disease and cancer in the Seven Countries Study. Arch
Intern Med 155, 381 – 386.
Heruth, D. P., Wetmore, L. A., Leyva, A., & Rothberg, P. G. (1995).
174
D.F. Birt et al. / Pharmacology & Therapeutics 90 (2001) 157–177
Influence of protein tyrosine phosphorylation on the expression of the
c-myc oncogene in cancer of the large bowel. J Cell Biochem 58,
83 – 94.
Hirano, T., Gotoh, M., & Oka, K. (1994). Natural flavonoids and lignans
are potent cytostatic agents against human leukemic HL-60 cells. Life
Sci 55, 1061 – 1069.
Hollman, P. C. H., & Katan, M. B. (1997). Absorption, metabolism and
health effects of dietary flavonoids in man. Biomed Pharmacother 51,
305 – 310.
Hollman, P. C. H., & Katan, M. B. (1998). Bioavailability and health effects
of dietary flavonols in man. Arch Toxicol Suppl 20, 237 – 248.
Hollman, P. C. H., De Vries, J. H. M., Van Leeuwen, S. D., Mengelers, M.
J. B., & Katan, M. B. (1995). Absorption of dietary quercetin glycosides and quercetin in healthy ileostomy volunteers. Am J Clin Nutr 62,
1276 – 1282.
Hollman, P. C. H., Van Trijp, J. M. P., Mengelers, M. J. B., De Vries, J. H. M.,
& Katan, M. B. (1997). Bioavailability of the dietary antioxidant flavonol
quercetin in man. Cancer Lett 114, 139 – 140.
Hollman, P. C. H., Bijsman, M. N., van Gameren, Y., Cnossen, E. P., de
Vries, J. H., & Katan, M. B. (1999). The sugar moiety is a major
determinant of the absorption of dietary flavonoid glycosides in man.
Free Radic Res 31, 569 – 573.
Hosokawa, N., Hosokawa, Y., Sakai, T., Yoshida, M., Marui, N., Nishino, H.,
Kawai, K., & Aoike, A. (1990). Inhibitory effect of quercetin on the
synthesis of a possibly cell-cycle-related 17-kDa protein, in human colon
cancer cells. Int J Cancer 45, 1119 – 1124.
Hsieh, C. Y., Santell, R. C., Haslam, S. Z., & Helferich, W. G. (1998).
Estrogenic effects of genistein on the growth of estrogen receptor-positive human breast cancer (MCF-7) cells in vitro and in vivo. Cancer
Res 58, 3833 – 3838.
Hu, J., Zhang, S., Jia, E., Wang, Q., Liu, S., Liu, Y., Wu, Y., & Cheng, Y.
(1988). Diet and cancer of the stomach: a case-control study in China.
Int J Cancer 41, 331 – 335.
Hudson, B. J. F., & Lewis, J. I. (1983). Polyhydroxy flavonoid antioxidants
for edible oils: structural criteria for activity. Food Chem 10, 47 – 51.
Hughes, D. A. (1999). Effects of dietary antioxidants on the immune function of middle-aged adults. Proc Nutr Soc 58, 79 – 84.
Husain, S. R., Cillard, J., & Cillard, P. (1987). Hydroxyl radical scavenging
activity of flavonoids. Phytochemistry 26, 2489 – 2491.
Hutchins, A. M., Slavin, J. L., & Lampe, J. W. (1995). Urinary isoflavonoid
phytoestrogen and lignan excretion after consumption of fermented and
unfermented soy products. J Am Diet Assoc 95, 545 – 551.
Kal, H. B., De Graaff, E., Van Berkel, A. H., & Goedoen, H. H. (1992).
Responses of experimental rat tumours and a mouse colon tumour to
flavone acetic acid. In Vivo 6, 73 – 76.
Kanazawa, K., Yamashita, T., Ashida, H., & Danno, G. (1998). Antimutagenicity of flavones and flavonols to heterocyclic amines by specific
and strong inhibition of the cytochrome P450 1A family. Biosci Biotechnol Biochem 62, 970 – 977.
Kandaswami, C., Perkins, E., Soloniuk, D. S., Drzewiecki, G., & Middleton,
E., Jr. (1991). Antiproliferative effects of citrus flavonoids on a human
squamous cell carcinoma in vitro. Cancer Lett 56, 147 – 152.
Karr, S. C., Lampe, J. W., Hutchins, A. M., & Slavin, J. L. (1997). Urinary
isoflavonoid excretion in humans is dose dependent at low to moderate
levels of soy-protein consumption. Am J Clin Nutr 66, 46 – 51.
Kawaii, S., Tomono, Y., Katase, E., Ogawa, K., & Yano, M. (1999). Antiproliferative activity of flavonoids on several cancer cell lines. Biosci
Biotechnol Biochem 63, 896 – 899.
Kawamori, T., Tanaka, T., Hara, A., Yamahara, J., & Mori, H. (1995).
Modifying effects of naturally occurring products on the development
of colonic aberrant crypt foci induced by azoxymethane in F344 rats.
Cancer Res 55, 1277 – 1282.
Kelly, G. E., Nelson, C., Waring, M. A., Joannou, G. E., & Reeder, A. Y.
(1993). Metabolites of dietary (soya) isoflavones in human urine. Clin
Chim Acta 223, 9 – 22.
Kennedy, A. R. (1995). The evidence for soybean products as cancer preventive agents. J Nutr 125, 733S – 743S.
King, R. A., & Bursill, D. B. (1998). Plasma and urinary kinetics of the
isoflavones daidzein and genistein after a single soy meal in humans.
Am J Clin Nutr 67, 867 – 872.
King, R. A., Broadbent, J. L., & Head, R. J. (1996). Absorption and excretion of the soy isoflavone genistein in rats. J Nutr 126, 176 – 182.
Knekt, P., Järvinen, R., Seppänen, R., Heliövaara, M., Teppo, L., Pukkala, E.,
& Aromaa, A. (1997). Dietary flavonoids and the risk of lung cancer and
other malignant neoplasms. Am J Epidemiol 146, 223 – 230.
Knight, D. C., & Eden, J. A. (1996). A review of the clinical effects of
phytoestrogens. Obstet Gynecol 87, 897 – 904.
Konoshima, T., Kokumai, M., Kozuka, M., Iinuma, M., Mizuno, M. Tanaka,
T., Mizuno, M., Tokuda, H., Nishino, H., & Iwashima, A. (1992). Studies
on inhibitors of skin tumor promotion. XI. Inhibitory effects of flavonoids
from Scutellaria baicalensis on Epstein – Barr virus activation and their
anti-tumor-promoting activities. Chem Pharm Bull (Tokyo) 40, 531 – 533.
Koo, L. C. (1988). Dietary habits and lung cancer risk among Chinese
females in Hong Kong who never smoked. Nutr Cancer 11, 155 – 172.
Kuo, M. L., & Yang, N. C. (1995). Reversion of v-H-ras-transformed NIH
3T3 cells by apigenin through inhibiting mitogen activated protein
kinase and its downstream oncogenes. Biochem Biophys Res Commun
212, 767 – 775.
Kuo, M.-L., Lin, J.-K., Huang, T.-S., & Yang, N.-C. (1994). Reversion of the
transformed phenotypes of v-H-ras NIH3T3 cells by flavonoids through
attenuating the content of phosphotyrosine. Cancer Lett 87, 91 – 97.
Kuo, S. M. (1996). Antiproliferative potency of structurally distinct dietary
flavonoids on human colon cancer cells. Cancer Lett 110, 41 – 48.
Kuo, S. M. (1997). Dietary flavonoid and cancer prevention: evidence and
potential mechanism. Crit Rev Oncog 8, 47 – 69.
Kyle, E., Neckers, L., Takimoto, C., Curt, G., & Bergan, R. (1997). Genistein-induced apoptosis of prostate cancer cells is preceded by a specific decrease in focal adhesion kinase activity. Mol Pharmacol 51,
193 – 200.
Lahiri-Chatterjee, M., Katiyar, S. K., Mohan, R. R., & Agarwal, R. (1999).
A flavonoid antioxidant, silymarin, affords exceptionally high protection against tumor promotion in the SENCAR mouse skin tumorigenesis model. Cancer Res 59, 622 – 632.
Lamartiniere, C. A., Moore, J., Holland, M., & Barnes, S. (1995). Neonatal
genistein chemoprevents mammary cancer. Proc Soc Exp Biol Med 208,
120 – 123.
Lamartiniere, C. A., Murrill, W. B., Manzolillo, P. A., Zhang, J. X.,
Barnes, S., Zhang, X. S., Wei, H. C., & Brown, N. M. (1998a). Genistein alters the ontogeny of mammary gland development and protects
against chemically-induced mammary cancer in rats. Proc Soc Exp Biol
Med 217, 358 – 364.
Lamartiniere, C. A., Zhang, J. X., & Cotroneo, M. S. (1998b). Genistein
studies in rats: potential for breast cancer prevention and reproductive
and developmental toxicity. Am J Clin Nutr 68, 1400S – 1405S.
Lampe, J. W., Karr, S. C., Hutchins, A. M., & Slavin, J. L. (1998). Urinary
equol excretion with a soy challenge: influence of habitual diet. Proc
Soc Exp Biol Med 217, 335 – 339.
Le Bail, J. C., Varnat, F., Nicolas, J. C., & Habrioux, G. (1998). Estrogenic
and antiproliferative activities on MCF-7 human breast cancer cells by
flavonoids. Cancer Lett 130, 209 – 216.
Lee, K. W., Wang, H. J., Murphy, P. A., & Hendrich, S. (1995). Soybean
isoflavone extract suppresses early but not later promotion of hepatocarcinogenesis by phenobarbital in female rat liver. Nutr Cancer 24,
267 – 278.
Lee, S. J., Choi, J. H., Son, K. H., Chang, H. W., Kang, S. S., & Kim, H. P.
(1995). Suppression of mouse lymphocyte proliferation in vitro by
naturally-occurring biflavonoids. Life Sci 57, 551 – 558.
Lee, S. K., Luyengi, L., Gerhäuser, C., Mar, W., Lee, K., Mehta, R. G.,
Kinghorn, A. D., & Pezzuto, J. M. (1999). Inhibitory effect of munetone, an isoflavonoid, on 12-O-tetradecanoylphorbol 13-acetate-induced
ornithine decarboxylase activity. Cancer Lett 136, 59 – 65.
Lepley, D. M., & Pelling, J. C. (1997). Induction of p21/WAF1 and G1 cellcycle arrest by the chemopreventive agent apigenin. Mol Carcinog 19,
74 – 82.
D.F. Birt et al. / Pharmacology & Therapeutics 90 (2001) 157–177
Lepley, D. M., Li, B. Y., Birt, D. F., & Pelling, J. C. (1996). The chemopreventive flavonoid apigenin induces G2/M arrest in keratinocytes.
Carcinogenesis 17, 2367 – 2375.
Lewis, N. G. (1993). Plant phenolics. In R. G. Alscher, & J. L. Hess (Eds.),
Antioxidants in Higher Plants ( pp. 135 – 169). Boca Raton: CRC Press.
Li, D. H., Yee, J. A., McGuire, M. H., Murphy, P. A., & Yan, L. (1999).
Soybean isoflavones reduce experimental metastasis in mice. J Nutr
129, 1075 – 1078.
Lian, F. R., Bhuiyan, M., Li, Y. W., Wall, N., Kraut, M., & Sarkar, F. H.
(1998). Genistein-induced G2-M arrest, p21WAF1 upregulation, and
apoptosis in a non-small-cell lung cancer cell line. Nutr Cancer 31,
184 – 191.
Lichtenstein, A. H. (1998). Soy protein, isoflavones and cardiovascular
disease risk. J Nutr 128, 1589 – 1592.
Lien, E. J., Ren, S., Bui, H. H., & Wang, R. (1999). Quantitative structure –
activity relationship analysis of phenolic antioxidants. Free Radic Biol
Med 26, 285 – 294.
Lowell, J. A., Parnes, H. L., & Blackburn, G. L. (1990). Dietary immunomodulation: beneficial effects on oncogenesis and tumor growth. Crit
Care Med 18, s145 – s148.
Lu, L. J., Grady, J. J., Marshall, M. V., Ramanujam, V. M., & Anderson, K.
E. (1995). Altered time course of urinary daidzein and genistein excretion during chronic soya diet in healthy male subjects. Nutr Cancer 24,
311 – 323.
Lu, L. J., Lin, S. N., Grady, J. J., Nagamani, M., & Anderson, K. E. (1997).
Altered kinetics and extent of urinary daidzein and genistein excretion
in women during chronic soya exposure. Nutr Cancer 26, 289 – 302.
Makela, S. I., Pylkkanen, L., Risto, S. S. S., & Adlercreutz, H. (1995).
Dietary soybean may be antiestrogenic in male mice. J Nutr 125,
437 – 445.
Makita, H., Tanaka, T., Fujitsuka, H., Tatematsu, N., Satoh, K., Hara, A., &
Mori, H. (1996). Chemoprevention of 4-nitraquinoline 1-oxide-induced
rat oral carcinogenesis by the dietary flavonoids chalcone, 2-hydroxychalcone, and quercetin. Cancer Res 56, 4904 – 4909.
Markaverich, B. M., Gregory, R. R., Alejandro, M. A., Clark, J. H., Johnson,
G. A., & Middledithc, B. S. (1988). Methyl p-hydroxyphenyllactate —
an inhibitor of cell growth and proliferation and an endogenous ligand
for nuclear type-II binding sites. J Biol Chem 263, 7203 – 7210.
Matsukawa, Y., Marui, N., Sakai, T., Satomi, Y., Yoshida, M., Matsumoto,
K., Nishino, H., & Aoike, A. (1993). Genistein arrests cell cycle progression at G2-M. Cancer Res 53, 1328 – 1331.
Messina, M., Descheemaker, K., & Erdman, J. W., Jr. (1998). The role of
soy in preventing and treating chronic disease. Am J Clin Nutr 68,
1329S (Proceedings of the Second International Symposium on the
Role of Soy in Preventing and Treating Chronic Disease held on September 15 – 18, 1996, and a satellite symposium held on September 19,
1996, in Brussels, Belgium — Preface).
Middleton, E., Jr. (1998). Effect of plant flavonoids on immune and inflammatory cell function. Adv Exp Med Biol 439, 175 – 182.
Middleton, E., & Kandaswami, C. (1992). Effects of flavonoids on immune
and inflammatory cell functions. Biochem Pharmacol 43, 1167 – 1179.
Miksicek, R. J. (1993). Commonly occurring plant flavonoids have estrogenic activity. Mol Pharmacol 44, 37 – 43.
Miksicek, R. J. (1995). Estrogenic flavonoids: structural requirements for
biological activity. Proc Soc Exp Biol Med 201, 44 – 50.
Mitchell, J. H., Gardner, P. T., McPhail, D. B., Morrice, P. C., Collins,
A. R., & Duthie, G. G. (1998). Antioxidant efficacy of phytoestrogens
in chemical and biological model systems. Arch Biochem Biophys 360,
142 – 148.
Mitsuoka, T. (1982). Recent trends in research on intestinal flora. Bifidobacteria Microflora 3, 3 – 24.
Moule, G. R., Braden, A. W. H., & Lamond, D. R. (1963). The significance
of oestrogens in pasture plants in relation to animal production. Anim
Breeding Abstr 31, 139 – 157.
Naik, H. R., Lehr, J. E., & Pienta, K. J. (1994). An in vitro and in vivo
study of antitumor effects of genistein on hormone refractory prostate
cancer. Anticancer Res 14, 2617 – 2620.
175
Nijhoff, W. A., Groen, G. M., & Peters, W. H. M. (1993). Induction of rat
hepatic and intestinal glutathione S-transferases and glutathione by dietary naturally occurring anticarcinogens. Int J Oncol 3, 1131 – 1139.
Nixon, J. E., Hendricks, J. D., Pawlowski, N. E., Pereira, C. B., Sinnhuber,
R. O., & Bailey, G. S. (1984). Inhibition of aflatoxin B1 carcinogenesis
in rainbow trout by flavone and indole compounds. Carcinogenesis 5,
615 – 619.
Noroozi, M., Angerson, W. J., & Lean, M. E. J. (1998). Effects of flavonoids and vitamin C on oxidative DNA damage to human lymphocytes.
Am J Clin Nutr 67, 1210 – 1218.
Omenn, G. S. (1995). What accounts for the association of vegetables and
fruits with lower incidence of cancers and coronary heart diseases. Ann
Epidemiol 5, 333 – 335.
Omenn, G. S., Goodman, G. E., Thornquist, M. D., Balmes, J., Cullen, M. R.,
Glass, A., Keogh, J. P., Meyskens, F. L., Jr., Valanis, B., Williams, J. H.,
Jr., Barnhart, S., & Hammar, S. (1996). Effects of a combination of beta
carotene and vitamin A on lung cancer and cardiovascular disease. N Engl
J Med 334, 1150 – 1155.
Onozawa, M., Kawamori, T., Baba, M., Fukuda, K., Toda, T., Sato, H.,
Ohtani, M., Akaza, H., Sugimura, T., & Wakabayashi, K. (1999). Effects
of a soybean isoflavone mixture on carcinogenesis in prostate and seminal vesicles of F344 rats. Jpn J Cancer Res (Amsterdam) 90, 393 – 398.
Pereira, M. A., Barnes, L. H., Rassman, V. L., Kelloff, G. V., & Steele, V. E.
(1994). Use of azoxymethane-induced foci of aberrant crypts in rat
colon to identify potential cancer chemopreventive agents. Carcinogenesis 15, 1049 – 1054.
Peterson, T. G., Ji, G. P., Kirk, M., Coward, L., Falany, C. N., & Barnes, S.
(1998). Metabolism of the isoflavones genistein and biochanin A in
human breast cancer cell lines. Am J Clin Nutr 68, 1505S – 1511S.
Piantelli, M., Rinelli, A., Macri, E., Maggiano, N., Larocca, L. M., Scerrati,
M., Roselli, R., Iacoangeli, M., Scambia, G., Capelli, A., & Ranelletti,
F. O. (1993). Type II estrogen binding sites and antiproliferative activity
of quercetin in human meningiomas. Cancer 71, 193 – 198.
Pierpoint, W. S. (1986). Flavonoids in the human diet. In V. Cody, E.
Middleton, Jr., & J. B. Harborne (Eds.), Progress in Clinical and
Biological Research (pp. 125 – 140). New York: Alan R. Liss.
Plaumann, B., Fritsche, M., Rimpler, H., Brandner, G., & Hess, R. D.
(1996). Flavonoids activate wild-type p53. Oncogene 13, 1605 – 1614.
Pollard, M., & Luckert, P. H. (1997). Influence of isoflavones in soy protein
isolates on development of induced prostate-related cancers in L – W
rats. Nutr Cancer 28, 41 – 45.
Potter, J. D., & Steinmetz, K. (1996). Vegetables, fruit and phytoestrogens
as preventive agents. IARC Sci Publ 139, 61 – 90.
Ratty, A. K., & Das, N. P. (1988). Effects of flavonoids on nonenzymatic
lipid peroxidation: structure – activity relationship. Biochem Med Metab
Biol 39, 69 – 79.
Renaud, S., & De Lorgeril, M. (1992). Wine, alcohol, platelets, and the
French paradox for coronary heart disease. Lancet 339, 1523 – 1526.
Robak, J., & Gryglewski, R. J. (1988). Flavonoids are scavengers of superoxide anion. Biochem Pharmacol 37, 83 – 88.
Rodgers, E. H., & Grant, M. H. (1998). The effect of the flavonoids, quercetin, myricetin and epicatechin on the growth and enzyme activities of
MCF7 human breast cancer cells. Chem Biol Interact 116, 213 – 228.
Setchell, K. D., Borriello, S. P., Hulme, P., Kirk, D. N., & Axelson, M.
(1984). Nonsteroidal estrogens of dietary origin: possible roles in hormone-dependent disease. Am J Clin Nutr 40, 569 – 578.
Severson, R. K., Nomura, A. M. Y., Grove, J. S., & Stemmermann, G. N.
(1989). A prospective study of demographics, diet, and prostate cancer
among men of Japanese ancestry in Hawaii. Cancer Res 49, 1857 – 1860.
Sfakianos, J., Coward, L., Kirk, M., & Barnes, S. (1997). Intestinal uptake
and biliary excretion of the isoflavone genistein in rats. J Nutr 127,
1260 – 1268.
Shahidi, F., Janitha, P. K., & Wanasundara, P. D. (1992). Phenolic antioxidants. Crit Rev Food Sci Nutr 32, 67 – 103.
Shutt, D. A., & Cox, R. I. (1972). Steroid and phyto-estrogen binding to
sheep uterine receptors in vitro. J Endocrinol 52, 299 – 310.
So, F. V., Guthrie, N., Chambers, A. F., Moussa, M., & Carroll, K. K.
176
D.F. Birt et al. / Pharmacology & Therapeutics 90 (2001) 157–177
(1996). Inhibition of human breast cancer cell proliferation and delay of
mammary tumorigenesis by flavonoids and citrus juices. Nutr Cancer
26, 167 – 181.
Sorensen, I. K., Kristiansen, E., Mortensen, A., Nicolaisen, G. M., Wijnandes, J. A. H., Van Kranen, H. J., & Van Kreijl, C. F. (1998). The
effect of soy isoflavones on the development of intestinal neoplasia in
ApcMin mouse. Cancer Lett 130, 217 – 225.
Spinozzi, F., Pagliacci, M. C., Miglioati, G., Moraca, R., Grignani, F.,
Riccardi, C., & Nicoletti, I. (1994). The natural tyrosine kinase inhibitor
genistein produces cell cycle arrest and apoptosis in Jukat T-leukemia
cells. Leuk Res 18, 431 – 439.
Steinmetz, K. A., & Potter, J. D. (1991a). Vegetables, fruit, and cancer. I.
Epidemiology. Cancer Causes Control 2, 325 – 357.
Steinmetz, K. A., & Potter, J. D. (1991b). Vegetables, fruit, and cancer. II.
Mechanisms. Cancer Causes Control 2, 427 – 442.
Steinmetz, K. A., Kushi, L. H., Bostick, R. M., Folsom, A. R., & Potter,
J. D. (1994). Vegetables, fruit, and colon cancer in the Iowa women’s
health study. Am J Epidemiol 139, 1 – 15.
Talalay, P. (1989). Mechanisms of induction of enzymes that protect against
chemical carcinogenesis. Adv Enzyme Regul 28, 237 – 250.
Talalay, P., Fahey, J. W., Holtzclaw, W. D., Prestera, T., & Zhang, Y. (1995).
Chemoprotection against cancer by phase 2 enzyme induction. Toxicol
Lett 82 – 83, 173 – 179.
Tanaka, T., Makita, H., Kawabata, K., Mori, H., Kakumoto, M., Satoh, K.,
Hara, A., Sumida, T., & Ogawa, H. (1997a). Chemoprevention of
azoxymethane-induced rat colon carcinogenesis by the naturally occurring flavonoids, diosmin and hesperidin. Carcinogenesis 18, 957 – 965.
Tanaka, T., Makita, H., Ohnishi, M., Mori, H., Satoh, K., Hara, A., Sumida,
T., Fukutani, K., & Ogawa, H. (1997b). Chemoprevention of 4-nitroquinoline 1-oxide-induced oral carcinogenesis in rats by flavonoids
diosmin and hesperidin, each alone and in combination. Cancer Res
57, 246 – 252.
Tew, B. Y., Xu, X., Wang, H.-J., Murphy, P. A., & Hendrich, S. (1996). A
diet high in wheat fiber suppresses the bioavailability of isoflavones in a
single meal fed to women. J Nutr 126, 871 – 877.
Thiagarajan, D. G., Bennink, M. R., Bourquin, L. D., & Kavas, F. A.
(1998). Prevention of precancerous colonic lesions in rats by soy flakes,
soy flour, genistein, and calcium. Am J Clin Nutr 68, 1394S – 1399S.
Torel, J., Cillard, J., & Cillard, P. (1986). Antioxidant activity of flavonoids
and reactivity with peroxy radical. Phytochemistry 25, 383 – 385.
Traganos, F., Ardelt, B., Halko, N., Bruno, S., & Darzynkiewicz, Z. (1992).
Effects of genistein on the growth and cell cycle progression of normal
human lymphocytes and human leukemic MOLT-4 and HL-60 cells.
Cancer Res 52, 6200 – 6208.
Verdeal, K., & Ryan, D. S. (1979). Naturally-occurring estrogens in plant
foodstuffs — a review. J Food Prot 42, 577 – 583.
Waladkhani, A. R., & Clemens, M. R. (1998). Effect of dietary phytochemicals on cancer development. Int J Med 1, 747 – 753.
Wang, G.-J. (1997). Human gut microfloral metabolism of soybean isoflavones. Master’s Thesis, Iowa State University.
Wang, H.-J., & Murphy, P. A. (1994). Isoflavone content of commercial
soybean foods. J Agric Food Chem 42, 1666 – 1673.
Wang, W., & Goodman, M. T. (1999). Antioxidant property of dietary
phenolic agents in a human LDL-oxidation ex vivo model: interaction
of protein binding activity. Nutr Res 19, 191 – 202.
Wang, W., & Higuchi, C. M. (1995). Induction of NAD(P)H:quinone reductase by vitamins A, E and C in Colo205 colon cancer cells. Cancer
Lett 98, 63 – 69.
Wang, W., & Higuchi, C. M. (2000). Dietary soy protein is associated with
reduced intestinal mucosal polyamine concentration in male Wistar rats.
J Nutr 130, 1815 – 1820.
Wang, W., Tanaka, Y., Han, Z., & Higuchi, C. M. (1995). Proliferative
response of mammary glandular tissues to formononetin. Nutr Cancer
23, 131 – 140.
Wang, W., Higuchi, C. M., & Zhang, R. (1997). Individual and combinatory effects of soy isoflavones on the in vitro potentiation of lymphocyte
activation. Nutr Cancer 29, 29 – 34.
Wang, W., Liu, L. Q., Higuchi, C. M., & Chen, H. (1998). Induction of
NADPH:quinone reductase by dietary phytoestrogens in colonic
Colo205 cells. Biochem Pharmacol 56, 189 – 195.
Wang, W., Heideman, L., Chung, C. S., Pelling, J. C., Koehler, K. J., &
Birt, D. F. (2000). Cell-cycle arrest at G2/M and growth inhibition
by apigenin in human colon carcinoma cell lines. Mol Carcinog 28,
102 – 110.
Watanabe, S., Yamaguchi, M., Sobue, T., Takahashi, T., Miura, T., Arai, Y.,
Mazur, W., Wahala, K., & Adlercreutz, H. (1998). Pharmacokinetics of
soybean isoflavones in plasma, urine and feces of men after injestion of
60 g baked soybean powder (Kinako). J Nutr 128, 1710 – 1715.
Watson, R. R. (1986). Immunological enhancement by fat-soluble vitamins,
minerals, and trace metals: a factor in cancer prevention. Cancer Detect
Prev 9, 67 – 77.
Wattenberg, L. W. (1992a). Inhibition of carcinogenesis by minor dietary
constituents. Cancer Res 52 (suppl.), 2085s – 2091s.
Wattenberg, L. W. (1992b). Chemoprevention of cancer by naturally occurring and synthetic compounds. In L. W. Wattenberg, M. Lipkin, C. W.
Kelloff, & G. J. Kelloff (Eds.), Cancer Chemoprevention ( pp. 19 – 39).
Boca Raton: CRC Press.
Wei, H., Tye, L., Bresnick, E., & Birt, D. F. (1990). Inhibitory effect of
apigenin, a plant flavonoid, on epidermal ornithine decarboxylase and
skin tumor promotion in mice. Cancer Res 50, 499 – 502.
Wei, H. C., Bowen, R., Zhang, X. S., & Lebwohl, M. (1998). Isoflavone
genistein inhibits the initiation and promotion of two-stage skin carcinogenesis in mice. Carcinogenesis 19, 1509 – 1514.
Wei, Y.-Q., Kariya, Y., Fukata, H., Teshigawara, K., & Uchida, A. (1994a).
Induction of apoptosis by quercetin: involvemnt of heat shock protein.
Cancer Res 54, 4952 – 4957.
Wei, Y., Zhao, X., Kariya, Y., Fukata, H., Teshigawara, K., & Uchida, A.
(1994b). Induction of apoptosis by quercetin: involvement of heat shock
protein. Cancer Res 54, 4952 – 4957.
Winter, J., Moore, L. H., Dowell, V. R., Jr., & Bokkenheuser, V. D. (1989).
C-ring cleavage of flavonoids by human intestinal bacteria. Appl Environ Microbiol 55, 1203 – 1208.
Wiseman, H. (1999). The bioavailability of non-nutrient plant factors: dietary flavonoids and phyto-estrogens. Proc Nutr Soc 58, 139 – 146.
Wu, A. H., Ziegler, R. G., Nomura, A. M. Y., West, D. W., Kolonel, L. N.,
Horn-Ross, P. L., Hoover, R. N., & Pike, M. C. (1998). Soy intake and
risk of breast cancer in Asians and Asian Americans. Am J Clin Nutr 68,
1437S – 1443S.
Xu, X., Wang, H., Murphy, P. A., Cook, L., & Hendrich, S. (1994). Daidzein is a more bioavailable soymilk isoflavone than is genistein in adult
women. J Nutr 124, 825 – 832.
Xu, X., Harris, K. S., Wang, H. J., Murphy, P. A., & Hendrich, S. (1995).
Bioavailability of soybean isoflavones depends upon gut microflora in
women. J Nutr 125, 2307 – 2315.
Xu, X., Wang, H.-J., Murphy, P. A., & Hendrich, S. (2000). Neither background diet choice nor type or soy food affect isoflavone bioavailability
in women. J Nutr 130, 798 – 801.
Yamanaka, N., Oda, O., & Nagao, S. (1997). Prooxidant activity of caffeic
acid, dietary non-flavonoid phenolic acid, on Cu(2+)-induced low density lipoprotein oxidation. FEBS Lett 405, 186 – 190.
Yoshida, M., Yamamoto, M., & Nikaido, T. (1992). Quercetin arrests human leukemic T-cells in late G1 phase of the cell cycle. Cancer Res 52,
6676 – 6681.
Zhang, J.-X., Hallmans, G., Landstrom, M., Bergh, A., Damber, J.-E.,
Aman, P., & Adlercreutz, H. (1997). Soy and rye diets inhibit the
development of Dunning R3327 prostatic adenocarcinoma in rats. Cancer Lett 114, 313 – 314.
Zhang, R., Li, Y., & Wang, W. (1997). Enhancement of immune function in
mice fed high doses of soy daidzein. Nutr Cancer 29, 24 – 28.
Zhang, Y., Song, T. T., Cunnick, J. E., Murphy, P. A., & Hendrich, S.
(1999a). Daidzein and genistein glucuronides in vitro are weakly estrogenic and activate human natural killer cells in nutritionally relevant
concentrations. J Nutr 129, 399 – 405.
Zhang, Y., Wang, G. J., Song, T. T., Murphy, P. A., & Hendrich, S. (1999b).
D.F. Birt et al. / Pharmacology & Therapeutics 90 (2001) 157–177
Urinary disposition of the soybean isoflavones daidzein, genistein and
glycitein differs among humans with moderate fecal isoflavone degradation activity. J Nutr 129, 957 – 962.
Zheng, W., Dai, Q., Custer, L. J., Shu, X. O., Wen, W. Q., Jin, F., & Franke,
A. A. (1999). Urinary excretion of isoflavonoids and the risk of breast
cancer. Cancer Epidemiol Biomarkers Prev 8, 35 – 40.
Zheng, Y. (2000). Ethnicity and diet habits: influence on isoflavone bioavailability in women. Master’s Thesis, Iowa State University.
Zhou, J. R., Mukherjee, P., Gugger, E. T., Tanaka, T., Blackbum, G. L., &
Clinton, S. K. (1998). Inhibition of murine bladder tumorigenesis by
177
soy isoflavones via alterations in the cell cycle, apoptosis, and angiogenesis. Cancer Res 58, 5231 – 5238.
Zi, X. L., Grasso, A. W., Kung, H. J., & Agarwal, R. (1998). A flavonoid
antioxidant, silymarin, inhibits activation of erbB1 signaling and induces cyclin-dependent kinase inhibitors, G1 arrest, and anticarcinogenic
effects in human prostate carcinoma DU145 cells. Cancer Res 58,
1920 – 1929.
Zwi, L. J., Baguley, B. C., Gavin, J. B., & Wilson, W. R. (1989). Blood
flow failure as a major determinant in the antitumor action of flavone
acetic acid. J Natl Cancer Inst 81, 1005 – 1013.