Toxicology in Vitro 20 (2006) 187 210
www.elsevier.com/locate/toxinvit
Mini-review
Dietary flavonoids: Effects on xenobiotic and carcinogen metabolism
*
Young Jin Moon, Xiaodong Wang, Marilyn E. Morris
Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, University at Buffalo, State University of New York,
517 Hochstetter Hall, Amherst, NY 14260-1200, United States
Received 1 May 2004; accepted 1 June 2005
Available online 11 November 2005
Abstract
Flavonoids are present in fruits, vegetables and beverages derived from plants (tea, red wine), and in many dietary supplements or
herbal remedies including Ginkgo Biloba, Soy Isoflavones, and Milk Thistle. Flavonoids have been described as health-promoting, dis-
ease-preventing dietary supplements, and have activity as cancer preventive agents. Additionally, they are extremely safe and associated
with low toxicity, making them excellent candidates for chemopreventive agents. The cancer protective effects of flavonoids have been
attributed to a wide variety of mechanisms, including modulating enzyme activities resulting in the decreased carcinogenicity of xeno-
biotics. This review focuses on the flavonoid effects on cytochrome P450 (CYP) enzymes involved in the activation of procarcinogens
and phase II enzymes, largely responsible for the detoxification of carcinogens.
A number of naturally occurring flavonoids have been shown to modulate the CYP450 system, including the induction of specific
CYP isozymes, and the activation or inhibition of these enzymes. Some flavonoids alter CYPs through binding to the aryl hydrocarbon
receptor (AhR), a ligand-activated transcription factor, acting as either AhR agonists or antagonists. Inhibition of CYP enzymes, includ-
ing CYP 1A1, 1A2, 2E1 and 3A4 by competitive or mechanism-based mechanisms also occurs. Flavones (chrysin, baicalein, and galan-
gin), flavanones (naringenin) and isoflavones (genistein, biocha\nin A) inhibit the activity of aromatase (CYP19), thus decreasing
estrogen biosynthesis and producing antiestrogenic effects, important in breast and prostate cancers. Activation of phase II detoxifying
enzymes, such as UDP-glucuronyl transferase, glutathione S-transferase, and quinone reductase by flavonoids results in the detoxifica-
tion of carcinogens and represents one mechanism of their anticarcinogenic effects. A number of flavonoids including fisetin, galangin,
quercetin, kaempferol, and genistein represent potent non-competitive inhibitors of sulfotransferase 1A1 (or P-PST); this may represent
an important mechanism for the chemoprevention of sulfation-induced carcinogenesis.
Importantly, the effects of flavonoids on enzymes are generally dependent on the concentrations of flavonoids present, and the dif-
ferent flavonoids ingested. Due to the low oral bioavailability of many flavonoids, the concentrations achieved in vivo following dietary
administration tend to be low, and may not reflect the concentrations tested under in vitro conditions; however, this may not be true
following the ingestion of herbal preparations when much higher plasma concentrations may be obtained. Effects will also vary with
the tissue distribution of enzymes, and with the species used in testing since differences between species in enzyme activities also can
be substantial. Additionally, in humans, marked interindividual variability in drug-metabolizing enzymes occurs as a result of genetic
and environmental factors. This variability in xenobiotic metabolizing enzymes and the effect of flavonoid ingestion on enzyme expres-
sion and activity can contribute to the varying susceptibility different individuals have to diseases such as cancer. As well, flavonoids may
also interact with chemotherapeutic drugs used in cancer treatment through the induction or inhibition of their metabolism.
Ó 2005 Elsevier Ltd. All rights reserved.
Keywords: Dietary flavonoids; Xenobiotic metabolism; CYP; Phase II enzymes; Carcinogen
*
Corresponding author. Tel.: [phone]x230; fax: [phone].
E-mail address: [e-mail] (M.E. Morris).
0887-2333/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tiv.2005.06.048
188 Y.J. Moon et al. / Toxicology in Vitro 20 (2006) 187 210
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188
2. Effect of bioflavonoids on cytochrome P450 (CYP450) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188
2.1. CYP1. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192
2.2. CYP2E. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197
2.3. CYP3A4. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197
2.4. CYP19 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199
3. Effect of bioflavonoids on phase II enzymes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200
3.1. UDP-glucuronyltransferase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200
3.2. Glutathione-S-transferase (GST) and quinone reductase (QR). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202
3.3. Sulfotransferases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203
4. Difficulties in the prediction of in vivo metabolic effects in humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204
5. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
1. Introduction The focus of this paper will be the effects of flavonoids
on cytochrome P450 (CYP) and phase II enzymes which
Flavonoids are part of a family of naturally occurring are key enzymes involved in the metabolism of xenobiotics.
polyphenolic compounds and represent one of the most Table 2 presents a summary of the flavonoid-induced alter-
prevalent classes of compounds in vegetables, nuts, fruits ations in enzyme expression and/or activity described in
and beverages such as coffee, tea, and red wine (Hollman this review.
and Katan, 1997) as well as medical herbs (e.g., Silybum
marianum, Alpina officinarum, Hypericum perforatum). 2. Effect of bioflavonoids on cytochrome P450 (CYP450)
The average total intake of flavonoids in the United States
was estimated to be 1 g/day (Kuhnau, 1976; Scalbert and Cytochrome P450 enzymes (phase I monooxygenase
Williamson, 2000), but recent studies have indicated that enzymes) are widely known for their role in the metabolism
the intake varies widely (Beecher, 2003). More than 8000 of drugs and other foreign compounds. Thus, modulation
compounds with a flavonoid structure have been identified. of this enzyme system can influence the metabolism of
The large number of compounds arises from the various xenobiotics, producing effects of pharmacological and tox-
combinations of multiple hydroxyl and methoxyl group icological importance. A number of naturally occurring
substituents on the basic flavonoid skeleton (Hodek flavonoids have been shown to modulate the CYP450 sys-
et al., 2002). The classes of flavonoids include chalcones, tem, including the induction of specific CYP isozymes, and
flavones, flavonols, flavanones, flavanols, anthocyanins the activation or inhibition of these enzymes (Wood et al.,
and isoflavones (Table 1). The flavonoid natural products 1986). Wattenberg et al. (1968) began investigating the
exert a wide range of biochemical and pharmacological effects of flavonoids on the CYP system over 36 years
properties, with one of the most investigated effects being ago, and a number of subsequent studies have shown differ-
their cancer preventive activities. The cancer protective ent modulatory effects of flavonoids on CYP activity
effects of flavonoids have been attributed to a wide variety in vitro and in vivo. Many carcinogens are metabolized
of mechanisms, including free radical scavenging, modify- by CYP enzymes to either biologically inactive metabolites
ing enzymes that activate or detoxify carcinogens, and or to chemically reactive electrophilic metabolites that
inhibiting the induction of the transcription factor activa- covalently bind to DNA producing carcinogenicity
tor protein-1 (AP-1) activity by tumor promoters (Caniv- (Fig. 1) (Conney, 2003). The reactive metabolites may
enc-Lavier et al., 1996; Shih et al., 2000). Flavonoids also undergo additional metabolism by phase I or II enzymes
have inhibitory effect on the activities of many enzymes, to inactive products. Therefore, induction of either phase
including b-glucuronidase (Kim et al., 1994), lipoxygenase I or phase II enzymes can result in increased detoxification
(Laughton et al., 1991; Schewe et al., 2002), cyclooxygen- of carcinogens (Conney, 2003). Since many chemical car-
ase (Laughton et al., 1991), inducible nitric oxide synthase cinogens are metabolized by CYP enzymes to both inac-
(Raso et al., 2001), monooxygenase (Siess et al., 1995), thy- tive, as well as to carcinogenic metabolites, the effects of
roid peroxidase (Doerge and Chang, 2002), xanthine oxi- inducers of these enzymes on the carcinogenicity of a chem-
dase (Sheu et al., 1998), mitochondrial succinoxidase and ical will depend on the inducerÕs effects on the different met-
NADH-oxidase (Hodnick et al., 1994), phosphodiesterase abolic pathways. In animal studies, it has been reported
(Picq et al., 1989), phospholipase A2(Gil et al., 1994), and that inducers of CYPs usually decrease the carcinogenicity
protein kinase (Cushman et al., 1991). of chemical carcinogens in vivo; this suggests that induc-
Y.J. Moon et al. / Toxicology in Vitro 20 (2006) 187 210 189
Table 1
The chemical structures and major food sources of the 6 major flavonoid subgroups
Structure Example Major food sources
Basic structure of flavonoids
Chalcone Hop chalcones (xanthohumol and Hops, beer
dehydrocyclo-xanthohumol hydrate)
Flavone Acacetin Parsley, thyme, celery, sweet red peppers,
Apigenin honey, propolis
Baicalein
Chrysin
Diosmetin
Luteolin
Tangeretin
Flavonol Galangin Onions, kale, broccoli, apples, cherries,
Kaempferol berries, tea, red wine
Morin
Myricetin
Quercetin
Flavanone Eriodictyol Citrus
Hesperetin
Homoeriodictyol
Naringenin
Flavanol Catechin Cocoa, green tea, chocolate, red wine,
Epicatechin hawthorn, bilberry, motherwort,
Proanthocyanidins and other herbs
Anthocyanin Cyanidins Cherries, grapes, berries, red cabbage
Pigmented
compounds
Isoflavone Biochanin A Red clover, alfalfa, peas, soy
Genistein and other legumes
Diadzein
Equol
Formononetin
190 Y.J. Moon et al. / Toxicology in Vitro 20 (2006) 187 210
Table 2
Summary of flavonoid-induced enzyme induction or inhibition ("induction, #inhibition, in vivo study)
*
Phase I Flavonoids Effects References
CYP1A1 Quercetin "mRNA expression and activity Ciolino et al. (1999)
Kaempferol #TCDD or B[a]P-induced transcription Kang et al. (1999)
Galangin #Activity Ciolino and Yeh (1999)
"mRNA expression Ciolino and Yeh (1999)
#TCDD or DMBA-induced transcription Ciolino and Yeh (1999)
Green tea extracts "Activity* Liu et al. (2003)
Theaflavins #Activity in the intestine* Catterall et al. (2003)
Xanthohumol #Activity Henderson et al. (2000)
Baicalein #DMBA-induced transcription Chan et al. (2002)
#Activity Chan et al. (2002)
Dimethoxyflavone #Activity Wen et al. (2005)
#B[a]P-induced mRNA and protein expression, and Activity Wen et al. (2005)
Chrysin "Activity Wen et al. (2005)
Diosmin "mRNA expression Ciolino et al. (1998)
Diosmetin "mRNA expression Ciolino et al. (1998)
#DMBA-induced transcription Ciolino et al. (1998)
#Activity Doostdar et al. (2000)
Acacetin #Activity Doostdar et al. (2000)
Flavone #Activity Zhai et al. (1998)
Hesperetin #Activity Doostdar et al. (2000)
Biochanin A #DMBA-induced transcription and activity Chan and Leung (2003)
Genistein #DMBA-induced transcription and activity Chan and Leung (2003)
20-Hydroxychalcone #mRNA expression and activity Wang et al. (2005)
CYP1A2 Quercetin #Activity Tsyrlov et al. (1994)
Biapigenin #Activity Obach (2000)
Galangin #Activity Zhai et al. (1998)
Green tea extracts #TPA-induced transcription Shih et al. (2000)
"Activity* Liu et al. (2003)
8-Prenylnaringenin #Activity Henderson et al. (2000)
Isoxanthohumol #Activity Henderson et al. (2000)
Diosmetin #Activity Doostdar et al. (2000)
Acacetin #Activity Doostdar et al. (2000)
Flavone #Activity Zhai et al. (1998)
Tangeretin #Activity Obermeier et al. (1995)
Naringin #Activity* Fuhr et al. (1993)
Genistein #Activity* Helsby et al. (1998)
#Activity* Chen et al. (2004)
Equol #Activity* Helsby et al. (1998)
Diadzein #Activity* Peng et al. (2003)
CYP1B1 Xanthohumol #Activity Henderson et al. (2000)
Baicalein #DMBA-induced transcription Chan et al. (2002)
Diosmetin #Activity Doostdar et al. (2000)
Acacetin #Activity Doostdar et al. (2000)
Hesperetin #Activity Doostdar et al. (2000)
Biochanin A #DMBA-induced transcription and activity Chan and Leung (2003)
Genistein #DMBA-induced transcription and activity Chan and Leung (2003)
Diadzein #Activity Roberts et al. (2004)
Formononetin #Activity Roberts et al. (2004)
20-hydroxychalcone #mRNA expression and activity Wang et al. (2005)
CYP2E1 Genistein #Activity* Helsby et al. (1998)
Equol #Activity* Helsby et al. (1998)
Theaflavins #Protein level* Catterall et al. (2003)
Silybin (=Silibinin, Silybinin) #Activity Zuber et al. (2002)
CYP3A4 Naringenin #Activity* Fuhr (1998)
Hyperforin #Activity Obach (2000)
"mRNA expression Zhou et al. (2003)
"Activity* Bray et al. (2002)
Biapigenin #Activity Obach (2000)
Quercetin #Activity Obach (2000)
Silymarin #Activity Venkataramanan et al. (2000)
Silybin #Activity (at high conc.) Beckmann-Knopp et al. (2000)
Y.J. Moon et al. / Toxicology in Vitro 20 (2006) 187 210 191
Table 2 (continued)
Phase I Flavonoids Effects References
"Activity (at low conc.)
Genistein #Activity Foster et al. (2003)
Diadzein #Activity Foster et al. (2003)
Glabridin #Activity Zhou et al. (2004)
Myricetin #Activity Ho et al. (2001)
CYP19 Biochanin A #Activity Kao et al. (1998)
#Activity Le Bail et al. (2000)
#Activity Almstrup et al. (2002)
Formononetin #Activity Almstrup et al. (2002)
Equol #Activity Pelissero et al. (1996)
Chrysin #Activity Kao et al. (1998)
#Activity Le Bail et al. (2000)
#Activity Almstrup et al. (2002)
#Activity* Gambelunghe et al. (2003)
Naringenin #Activity Kao et al. (1998)
#Activity Le Bail et al. (2000)
#Activity Almstrup et al. (2002)
Flavone #Activity Le Bail et al. (2000)
Apigenin #Activity Le Bail et al. (2000)
Bicalein #Activity Kao et al. (1998)
Galangin #Activity Kao et al. (1998)
Phase II
UGT1A1 Chrysin "mRNA expression Walle et al. (2000)
"activity Galijatovic et al. (2001)
Galangin "mRNA expression Walle and Walle (2002)
Isorhamnetin "mRNA expression Walle and Walle (2002)
Tangeretin #Activity Williams et al. (2002)
Naringenin #Activity Williams et al. (2002)
Flavone #Activity Williams et al. (2002)
Quercetin #Activity Williams et al. (2002)
UGT Flavone "Activity* Brouard et al. (1988)
"Activity* Canivenc-Lavier et al. (1996)
"Activity* van der Logt et al. (2003)
Quercetin "Activity* van der Logt et al. (2003)
"Activity Sun et al. (1998)
Green tea "Activity* Bu-Abbas et al. (1995)
"Activity* Sohn et al. (1994)
Biochanin A "Activity Sun et al. (1998)
Formononetin "Activity Sun et al. (1998)
Genistein "Activity Sun et al. (1998)
Diadzein "Activity Sun et al. (1998)
Naringenin "Activity Sun et al. (1998)
Galangin "Activity Sun et al. (1998)
Kaempferol "Activity Sun et al. (1998)
GST Genistein "or #mRNA expression Ansell et al. (2004)
(cell type specific)
"Activity* Appelt and Reicks (1999)
Diadzein "Activity* Appelt and Reicks (1999)
Genistein + Diadzein Prevent TPA-downregulated Activity* Sharma and Sultana (2004)
Flavone "Activity* Nijhoff et al. (1995)
Green tea extract "Activity* Maliakal et al. (2001)
Morin "Activity* Tanaka et al. (1999)
"Activity* Kawabata et al. (1999)
Silymarin "Activity* Kohno et al. (2002)
GSTA2 Flavone "mRNA expression Kang et al. (2003)
20-amino-30 methoxyflavone "mRNA expression Kang et al. (2003)
GSTP1-1 Quercetin #Activity van Zanden et al. (2003)
QR Tephropurpurin "Activity Chang et al. (1997)
Xanthohumol "Activity Miranda et al. (2000)
(continued on next page)
192 Y.J. Moon et al. / Toxicology in Vitro 20 (2006) 187 210
Table 2 (continued)
Phase II Flavonoids Effects References
Morin "Activity* Tanaka et al. (1999)
"Activity* Kawabata et al. (1999)
Pinostrobin "Activity Fahey and Stephenson (2002)
Kaempferol "Activity Uda et al. (1997)
Galangin "Activity Uda et al. (1997)
Quercetin "Activity Uda et al. (1997)
Silymarin "Activity* Kohno et al. (2002)
Genistein "Activity Yannai et al. (1998)
"Activity and mRNA expression Wang et al. (1998)
"Activity* Appelt and Reicks (1999)
Diadzein "Activity Yannai et al. (1998)
"Activity* Appelt and Reicks (1999)
Flavone "Activity* Siess et al., 1996
SULT1A1 Fisetin #Activity Eaton et al. (1996)
(P-PST or TS-PST) Galangin #Activity Eaton et al. (1996)
Quercetin #Activity Eaton et al. (1996)
#Activity Ghazali and Waring (1999)
#Activity Harris et al. (2004)
Catechin #Activity Ghazali and Waring (1999)
Equol #Activity Ghazali and Waring (1999)
Flavone #Activity Ghazali and Waring (1999)
Myricetin #Activity Eaton et al. (1996)
Kaempferol #Activity Eaton et al. (1996)
Chrysin #Activity Eaton et al. (1996)
Apigenin #Activity Eaton et al. (1996)
Genistein #Activity Eaton et al. (1996)
#Activity Ghazali and Waring (1999)
#Activity Harris et al. (2004)
Diadzein #Activity Harris et al. (2004)
#Activity Ghazali and Waring (1999)
SULT1A3 Baicalein #Activity Harris et al. (2004)
SULT1E1 Equol #Activity Harris et al. (2004)
Quercetin #Activity Otake et al. (2000)
#Activity Ohkimoto et al. (2004)
Genistein #Activity Ohkimoto et al. (2004)
Diadzein #Activity Ohkimoto et al. (2004)
#Activity Wong and Keung (1997)
tion of Phase I and II detoxification pathways may occur to CYP1A2 plays a role in human tobacco-related cancers
a greater extent than induction of CYPs involved in the (Smith et al., 1996). CYP1A1 is poorly expressed in human
formation of carcinogenic metabolites (Fig. 1) (Conney, liver although its synthesis can be markedly induced in
2003). many extrahepatic tissues, notably the lungs (Rendic and
Di Carlo, 1997). In contrast, CYP1A2 is expressed princi-
2.1. CYP1 pally in the liver (Rendic and Di Carlo, 1997). CYP1B1
is an extrahepatic estradiol 4-hydroxylase that activates
The CYP1 family consists of 1A1, 1A2, and 1B1 mem- procarcinogens and elevated levels have been associated
bers that are capable of activating procarcinogens. with estrogen carcinogenesis (Jefcoate et al., 2000). Normal
CYP1A1 and 1B1 are both involved in the biotransforma- human breast and human breast tumor tissues are known
tion of polycyclic aromatic hydrocarbons (PAHs), a class to express CYP1B1, producing carcinogenic 4-hydroxy
of ubiquitous environmental chemicals, to carcinogenic estrogen. Inhibition of CYP1B1 affects the production of
metabolites (Guengerich and Shimada, 1991). This process mutagenic estrogen 3,4-catechols (Roberts et al., 2004).
is believed to contribute to pulmonary carcinogenesis, Xenobiotic responsive elements (XRE) are cis-acting
because increased lung CYP1A1 expression and activity enhancer elements located in the promoter regions of xeno-
are associated with a high risk of lung cancer (McLemore biotic responsive genes, which include genes encoding for
et al., 1990). High CYP1A1 activity is also associated with CYP1A1 and 1B1. The expression of these xenobiotic
colorectal cancer (Sivaraman et al., 1994). CYP1A2 mainly responsive genes can be regulated through pathway involv-
metabolizes important drugs such as phenacetin, theophyl- ing aryl hydrocarbon receptor (AhR), which is a cytosolic
line, caffeine, imipramine, and propranolol (Brosen, 1995), protein that can be activated by PAH. The activated
and also activates some procarcinogens to carcinogens. AhR then translocates to the nucleus, dimerizes with
Y.J. Moon et al. / Toxicology in Vitro 20 (2006) 187 210 193
Fig. 1. Flavonoids that block or suppress multistage carcinogenesis. Carcinogenesis is initiated with the transformation of the normal cell into a mutant
cell. These cells undergo tumor promotion into benign tumor cells, which progress to malignant cells. Flavonoids can interfere with different steps of this
process. Some flavonoids (for example, kaempferol, diosmetin, theaflavin, and biochanin A) can inhibit the metabolic activation of the procarcinogens to
their ultimate electrophilic species by phase I enzymes (predominantly CYPs), or their subsequent interaction with DNA. Therefore these agents block
tumor initiation (blocking agents). Alternatively, dietary flavonoids (for example, naringenin, quercetin, biochanin A, and prenylchalcones) can stimulate
the detoxification of carcinogens by inducing phase II enzymes, leading to their elimination from the body. Flavonoids such as genistein and EGCG
suppress the later steps (promotion and progression) of multistage carcinogenesis (suppressing agents) by affecting cell cycle, angiogenesis, invasion, and
apoptosis. Adapted from (Chen and Kong, 2004).
AhR nuclear translocator (ARNT), and interacts with (Fig. 2D) dependent increase in CYP1A1 enzyme activity
XRE (Kronenberg et al., 2000). Induction of CYP1A by in MCF-7 cells (Ciolino et al., 1999). Kaempferol, by itself,
flavonoids proceeds by various mechanisms, including the does not affect CYP1A1 expression (Fig. 2A), but it can
direct stimulation of gene expression via specific receptor(s) interact with the AhR, and act as an antagonist of TCDD-
and/or CYP protein, or mRNA stabilization (Lin and Lu, induced CYP1A1 transcription (Ciolino et al., 1999).
1998; Shih et al., 2000). Some flavonoids induce CYPs Despite the structural similarity between quercetin and
through binding to AhR, a ligand-activated transcription kaempferol, their differential effects might be due to the
factor, by acting in a similar way as 2,3,7,8-tetrachlo- absence of an additional hydroxy group on the B-ring of
rodibenzo-p-dioxin (TCDD) (Kohn et al., 2001). Gener- kaempferol (Fig. 3), preventing it from achieving an optimal
ally, substrates for AhR are planar aromatic compounds fit into the binding site on AhR to produce transcriptional
with few bulky substituent groups. That might partly activation. The binding of kaempferol may block the bind-
explain the activity of flavonoids, which have similar pla- ing of AhR ligands, and thus inhibit the activity of other
nar structures as AhR (Hodek et al., 2002; Kohn et al., ligands such as TCDD (Ciolino et al., 1999). The importance
2001; MacDonald et al., 2001). Other flavonoids have been of the ortho-orientation of the hydroxyl group on the B ring
shown to directly inhibit CYP1A1 activty (Doostdar et al., was tested by comparing the effects of quercetin with a 30,40-
2000; Tsyrlov et al., 1994; Wen et al., 2005), commonly substitution pattern and morin (20,40-substitution pattern)
demonstrated to be a competitive-type of inhibition, and (Tsyrlov et al., 1994). 7-Methoxyresorufin-O-dealkylation
to affect CYP1A1 transcription (Ciolino et al., 1999; Kang (MROD) activities of mouse c-DNA-expressed CYP1A1
et al., 1999). and 1A2, and human CYP1A2 were more effectively inhib-
The most abundant flavonoids, flavonols quercetin and ited with quercetin than with morin (Tsyrlov et al., 1994).
kaempferol, are both dietary ligands of the AhR, but they Orientation of the methyl group can also influence inhibition
exert different effects on CYP1A1 expression (Ciolino (Tsyrlov et al., 1994). Quercetin, a component in St. JohnÕs
et al., 1999). Treatment of MCF-7 cells with quercetin wort inhibits CYP1A2 (Obach, 2000). St. JohnÕs wort
resulted in a concentration- (Fig. 2A) and time (Fig. 2B) (Hypericum perforatum) extracts are commercially available
dependent increase in the amount of CYP1A1 mRNA. preparations used in the treatment of depression. Quercetin
The activity of enzyme CYP1A1 was measured by a 7-eth- inhibits phenacetin O-deethylase activity (mediated by
oxyresorufin-O-dealkylation (EROD) assay. The increase CYP1A2) in a recombinant CYP1A2 enzyme preparation
in EROD activity followed the increase in CYP1A1 mRNA. with an IC50 value of 7.5 lM. Another flavonoid in St.
Quercetin also causes a concentration- (Fig. 2C) and time- JohnÕs wort, I3,II8-biapigenin was also shown to be a
194 Y.J. Moon et al. / Toxicology in Vitro 20 (2006) 187 210
Fig. 2. Effect of quercetin or kaempferol on expression of mRNA CYP1A1 and CYP1A1 enzyme activity. (A) Concentration response of CYP1A1
mRNA to quercetin (hatched bars) and kaempferol (open bars). MCF-7 cells were treated with quercetin the indicated concentration of quercetin. The
amount of CYP1A1 was normalized to the GPDH level. nd, not determined. (B) Time course of CYP1A1 mRNA increase caused by quercetin. MCF-7
cells were treated with 0.5 lM quercetin for the times indicated. The amount of CYP1A1 mRNA was normalized to GPDH levels. (C) The activity of
CYP1A1 in intact MCF-7 cells was determined by EROD assay. Cells were treated with the indicated concentrations of quercetin (j) or kaempferol ( )
for 48 h. (D) Cells were treated with 5 lM quercetin for the times indicated. Reproduced from (Ciolino et al., 1999) with permission from the Biochemical
Society.
potent, competitive inhibitor of CYP1A2 activity (IC50 = and Yeh, 1999). Similarly, quercetin causes an increase in
3.7 lM) (Obach, 2000). the level of CYP1A1 mRNA (Ciolino et al., 1999), whereas
Galangin, a flavonol found in honey, is a potent inhibi- it significantly inhibits benzo(a)pyrene (B[a]P)-induced
tor of CYP1A1 activity, as measured by inhibition of CYP1A1 mRNA and protein expression in human Hep
EROD activity, in intact cells and in microsomes isolated G2 cells (Kang et al., 1999). Galangin is a very potent
from dimethylbenz[a]anthracene (DMBA) treated cells CYP1A2 inhibitor, too (Zhai et al., 1998). It showed the
(Ciolino and Yeh, 1999). The effect is dose-dependent. Gal- mixed-type inhibition, indicating that this compound can
angin is a non-competitive inhibitor of CYP1A1 activity compete for substrate binding at the active site and also
(Ciolino and Yeh, 1999). Galangin increased the level of may bind to a region that does not participate directly in
CYP1A1 mRNA, indicating that it may be an agonist of substrate binding (Zhai et al., 1998).
the AhR, but it inhibited the induction of CYP1A1 mRNA Activator protein-1 (AP-1) may be involved in the regu-
by DMBA or by 2,3,5,7- TCDD (Ciolino and Yeh, 1999). lation of human CYP1A2 which contains two AP-1 bind-
Galangin also inhibited the DMBA- or TCDD-induced ing sites (Shih et al., 2000). Shih et al. (2000) developed a
transcription of a reporter vector containing the CYP1A1 cell line T2Luc, which is a HepG2-derived cell line stably
promoter. Thus, galangin is a potent inhibitor of DMBA integrated with a region of the human CYP1A2 50-flanking
metabolism and an agonist/antagonist of the AhR (Ciolino gene containing two AP-1 binding sites linked to the thymi-
Y.J. Moon et al. / Toxicology in Vitro 20 (2006) 187 210 195
nols 833 mg/kg/day, intragastrically, for 6 months) groups
were significantly increased (Liu et al., 2003).
Theaflavins in black tea are catechins generated by oxi-
dation of flavanols during the fermentation of fresh tea leaf
(Graham, 1992). It has been shown to have antiprolifera-
tive and anticarcinogenic activities (Yang et al., 1997).
Male rats received intragastric administration of theaflav-
ins (20 mg/kg) for four weeks, and the liver and intestine
mucosa samples were obtained. Theaflavin treatment
markedly suppressed the CYP1A1 EROD activity in the
intestine (Catterall et al., 2003). However, there was no
effect on any CYP activity in the liver (Catterall et al.,
2003); these results contrast with those from another study
where theaflavins suppressed CYP1A1 activity in hepatic
cell cultures (Feng et al., 2002). This discrepancy is pro-
bably due to the poor bioavailability of theaflavins as a
result of poor absorption and/or extensive pre-systemic
metabolism.
Henderson et al. (2000) reported the in vitro inhibition
of cDNA-expressed human CYP1A1, CYP1B1, and
CYP1A2 by flavonoids from hops (Humulus lupulus) (Hen-
derson et al., 2000). At 10 lM, the prenylated chalcone,
xanthohumol, almost completely inhibited the EROD
activity of CYP1A1. At the same concentration, other
hop flavonoids decreased the EROD activity by 27.0
90.8%. At 10 lM, xanthohumol completely eliminated
Fig. 3. Structures of quercetin, kaempferol and 2,3,7,8-tetrachlo-
CYP1B1 EROD activity, whereas the other hop flavonoids
rodibenzo-p-dioxin (TCDD).
showed varying degrees of inhibitory action ranging from
1.8% to 99.3% (Henderson et al., 2000). The most effective
dine kinase promoter-driven firefly luciferase reporter gene.
inhibitors of CYP1A2 acetanilide 4-hydroxylase activity
Green tea extracts (GTEs) inhibited the phorbol 12-o- were the two prenylated flavonoids, 8-prenylnaringenin
tetradecanoate 13-acetate (TPA)-induced AP-1 transcrip- and isoxanthohumol, which produced >90% inhibition
tional activation of a human CYP1A2 enhancer element,
when added at concentrations of 10 lM. CYP1A2 meta-
while quercetin enhanced this activity. Green tea (Camellia
bolism of the carcinogen aflatoxin B1 was also inhibited
sinensis) and its extracts are rich source of catechins, a class
by isoxanthohumol and 8-prenylnaringenin as shown by
of flavonoids (Shih et al., 2000). Catechins are the main
decreased appearance of dihydrodiols and aflatoxin M1
compounds in green tea; they consist of ( )-epicatechin,
as analysed by HPLC (Henderson et al., 2000).
( )-epicatechin-3-gallate (ECG), ( )-epigallocatechin,
Baicalein, a flavone extracted from the root of the Scu-
and ( )-epigallocatechin-3-gallate (EGCG) (Graham,
tellaria species, is a strong competitive inhibitor of EROD
1992). The mechanism of inhibition may be due to changes
activity induced by DMBA in MCF-7 cells (Chan et al.,
in the composition of the AP-1 complex upon treatment of
2002). Baicalein can reduce the CYP1A1/1B1 mRNA
cells with GTEs. An important and unexpected finding is
expression induced by DMBA, and the effect on mRNA
that GTEs themselves increased binding of nuclear pro- abundance of CYP1A1 was greater than that of CYP1B1.
teins to the AP-1 site to the same extent as TPA, yet did
An XRE-luciferase gene reporter assay also indicated that
not activate transcription of the luciferase reporter gene.
AhR transactivation was suppressed (Chan et al., 2002).
Unlike TPA, which induces AP-1 binding as early as 6 h
5,7-Dimethoxyflavone (DMF) is a major constituent of
after its addition, GTE-induced AP-1 binding did not
the leaves of a Malaysian Piper species. DMF reduced
occur until after 6 h, suggesting that other upstream signal- CYP1A1 EROD activity of the HepG2 cells virtually down
ing pathways may also be targeted by this dietary agent
to zero (Wen et al., 2005). It also inhibited benzo(a)pyrene
(data not shown) (Shih et al., 2000). In some in vitro exper- (B[a]P)-induced DNA binding (Wen et al., 2005). B[a]P, a
iments, tea polyphenols had an inhibitory effect on micro- major PAH procarcinogen, induces CYP1A1 protein and
somal CYP enzyme system (Mukhtar et al., 1992; Wang
its catalytic activity as well as CYP1A1 mRNA; DMF
et al., 1988). In contrast, long-term consumption of green
clearly inhibits this transcriptional activation by decreasing
tea increases CYP1A1 and 1A2 activities in rats (Liu
CYP1A1 catalytic activity as well as protein and mRNA
et al., 2003). The contents of CYPs (measured by the
expression. DMF can also directly inhibit CYP1A1 protein
method of Omura and Sato (Heffernan and Winston,
catalytic activity, as determined with recombinant protein
2000)) in the livers of male rats in high dose (tea polyphe- (Wen et al., 2005). It is remarkable that two so seemingly
196 Y.J. Moon et al. / Toxicology in Vitro 20 (2006) 187 210
similar compounds as DMF and chrysin can have such dif-
ferent effects, i.e. the 5,7-dimethoxy compound is a potent
inhibitor of the CYP1A1 protein whereas the 5,7-dihy-
droxy compound is a potent inducer (Wen et al., 2005).
Thus, methylation of flavonoids seems to be an important
feature determining enzyme inhibitory properties.
The flavone diosmetin is ingested (as its glucoside, dios-
min) in the commercially available drug ÔDaflon 500 mgÕ
(90% diosmin and 10% hesperetin). Both diosmin and dios-
metin are agonists of the AhR, causing a dose-dependent
increase in expression of CYP1A1 mRNA in MCF-7
human breast epithelial cancer cells (Ciolino et al., 1998);
however, diosmetin, but not diosmin, inhibits CYP1A1
activity in a non-competitive manner in microsomes iso-
lated from 7,12-dimethylbenz-[a]anthracene (DMBA) trea-
ted cells, as assayed by EROD activity (Ciolino et al.,
1998).
Doostdar and co-workers (Doostdar et al., 2000) dem- Fig. 4. Lineweaver-Burk plot of EROD activity of lymphoblastoid
expressed human CYP1B1 (3.7 pmol) without hesperetin (d) and in
onstrated that six common flavonoids present in citrus
reactions containing 0.01 lM(j) or 0.02 lM hesperetin (m). Reproduced
juices at 70 200 mg/L are potent inhibitors of CYP1A1,
from (Doostdar et al., 2000), with permission from Elsevier Science Inc.
1A2 and 1B1 EROD activity in vitro (Doostdar et al.,
2000). The flavones acacetin and diosmetin were more
potent inhibitors than the flavanones eriodictyol, hespere- an effective inhibitor of recombinant human CYP1A1
and CYP1B1 with Ki values of 15.35 and 0.68 lM, respec-
tin, homoeriodictyol and naringenin. This is probably
tively (Chan and Leung, 2003). Extrahepatic CYP1B1
due to the presence of the reduced 2,3-bond in the C ring
catalyzes the O-demethylation of biochanin A and formo-
of the structure in the flavones (Table 1) (Dai et al.,
1998). Potent inhibition of CYP1A1 and 1A2 enzyme activ- nonetin to produce genistein and daidzein, respectively,
which inhibit CYP1B1 (Roberts et al., 2004). Inhibition
ity by flavone has also been reported in other studies (Lee
of CYP1B1 EROD activity by genistein was primarily
et al., 1994; Zhai et al., 1998). Flavone inhibits CYP1A1
and CYP1A2, with a 2-fold greater potency toward the lat- non-competitive (Ki of 1.9 lM), and daidzein exhibited
mixed, but predominantly non-competitive inhibition of
ter (Zhai et al., 1998).
CYP1B1 EROD activity (Ki of 3.7 lM) (Roberts et al.,
Tangeretin, a polymethoxylated flavone present in large
2004). Therefore, biochanin A and/or formononetin may
amounts in citrus fruits, is a potent inhibitor of CYP1A2
EROD activity, with IC50 of 0.8 lM in rat liver micro- exert anticarcinogenic effects directly by acting as compet-
itive substrates for CYP1B1 or indirectly through their
somes and 16 lM in human microsomes (Obermeier
et al., 1995). The inhibition was competitive with a inhibi- metabolites daidzein and genistein, which inhibit CYP1B1
(Roberts et al., 2004).
tion constant (Ki) value of 68 nM (Obermeier et al., 1995).
Genistein and equol inhibit CYP1A2 in liver micro-
The flavanone hesperetin is a selective substrate of
somes from b-naphthoflavone-induced mice with IC50 val-
human CYP1A1 and CYP1B1 in the lymphoblastoid cell
ues of 5.6 mM and 1.7 mM, respectively (Helsby et al.,
line AHH-1, and it is a competitive inhibitor of CYP1B1
1998). Using human CYP1A2 from a specific expression
(Fig. 4) (Doostdar et al., 2000). Hesperidin, the glycoside
system, non-competitive inhibition was seen with both iso-
of hesperetin, is the major flavonoid in orange juice. In vivo
studies have demonstrated that grapefruit juice consump- flavones. This inhibition offers a possible explanation for
tion increased the plasma half-lives of drugs such as caf- the chemopreventive effects of genistein in animals, but
inhibition of CYP1A2 is not likely to be achieved from
feine. This effect was attributed to inhibition of CYP1A2
the concentration of genistein present in the human diet
by naringin, the major flavonone in grapefruit juice (Fuhr
(Helsby et al., 1998).
et al., 1993).
Biochanin A, the red clover (Trifolium pretense) isoflav- Dietary soy isoflavone containing 155 mg/g of genistein,
one (Chan et al., 2003), and the soybean isoflavone geni- 127 mg/g of daidzein, and other minor isoflavones had no
effect on the hepatic mRNA abundance of CYP1A1 and
stein (Chan and Leung, 2003) are effective inhibitors of
DMBA-induced DNA damage in MCF-7 cells by inhibit- 1A2 in rats, determined by real-time quantitative RT-
PCR (Kishida et al., 2004). This indicates that dietary iso-
ing CYP1A1 and CYP1B1. Both isoflavones could reduce
flavones do not induce CYPs in either the transcriptional
xenobiotic-induced CYP1A1 and 1B1 mRNA expression
step or through post-transcriptional mRNA stabilization
through interference with XRE-dependent transactivation.
(Kishida et al., 2004). Another in vivo study has reported
Enzyme kinetic studies also indicated that biochanin A
that genistein and equol only affected the protein content
inhibits CYP1A1 and 1B1 with Ki values of 4.00 and
or activity of CYP1A1 and 1A2 following the administra-
0.59 lM, respectively (Chan et al., 2003), and genistein is
Y.J. Moon et al. / Toxicology in Vitro 20 (2006) 187 210 197
tion of daily intraperitoneal injections to mice, when doses medicine as an antiseptic and antiulcerative remedy. The
of 40 mg/kg or higher of these isoflavones were adminis- inhibitory potencies decreased in the following order:
tered (Helsby et al., 1997). galangin > 7-hydroxyflavone > quercetin, chrysin, 2-
Daidzein, a principal isoflavone in soybean, can inhibit hydroxychalcone > 4,6-dihydroxychalcone > homoisoflav-
CYP1A2 activity and alter the pharmacokinetics of the- anes (Machala et al., 2001).
ophylline in healthy volunteers (Peng et al., 2003). Theoph-
ylline is a bronchodilator with a narrow therapeutic index 2.2. CYP2E
(5 20 mg/L), and it is primarily eliminated by hepatic
metabolism mediated by CYP1A2. In the study by Peng CYP2E1, an ethanol-inducible enzyme, is important in
et al. (2003), a single dose of 100 mg theophylline was taken the field of toxicology and carcinogenesis, and it also has
on day 3. Thereafter, one group received 200 mg daidzein a role in drug metabolism (Guengerich et al., 1991). For
twice daily for 10 days, and the other group received pla- example, following an overdose of acetaminophen
cebo. On day 12, the test group received 200 mg daidzein CYP2E1 converts acetaminophen to toxic quinones, which
with 100 mg theophylline. Comparing the kinetic parame- is responsible for the initiation of centrilobular liver toxic-
ters of theophylline determined on day 1 (without co-med- ity (Lindros et al., 1990). The activity of CYP2E1 is known
ication) with those determined on day 12 (10-day daidzein), to be mainly regulated by post-transcrpitional protein sta-
the AUC, Cmax, and t1/2 were significantly increased (Peng bilization, but the contribution of the transcriptional step is
et al., 2003). also significant (Novak and Woodcroft, 2000).
Genistein as well as combination of three (genistein, Genistein and equol, isoflavones in soy products, inhib-
daidzein, and glycitein) to five (plus biochanin A and for- ited p-nitrophenol (CYP2E1 substrate) metabolism in liver
mononetin) isoflavones inhibited tamoxifen a-hydroxyl- microsomes from acetone-induced mice with IC50 values of
ation in female rat liver microsomes in vitro via approximately 10 mM and 560 lM, respecively (Helsby
inhibition of CYP1A2 (Chen et al., 2004). The inhibition et al., 1998). The 5-hydroxyl group and the 2,3-double
of a-hydroxylation by genistein was mixed-type with a Ki bond as well as hydroxyl groups in the flavonoid B ring
value of 10.6 lM(Chen et al., 2004). a-Hydroxytamoxifen may be essential for inhibition of aryl hydroxylation by
and its sulfate conjugate are thought to be responsible for CYP2E1 (Helsby et al., 1998).
DNA adduct formation (Umemoto et al., 2001). Thus, gen- Theaflavins, catechins in black tea, decreased the protein
istein and its isoflavone analogs have the potential to level of CYP2E1 in rat intestinal microsomes after oral
decrease the side effects of tamoxifen through metabolic intake for four weeks (Catterall et al., 2003). Theaflavins
interactions that inhibit the formation of a-hydroxylation have been shown to antagonize the carcinogenicity of
(Chen et al., 2004). nitrosamines in mice (Shukla and Taneja, 2002) and to
Chalcones belong to the flavonoid family and, naturally have antimutagenic activity (Apostolides et al., 1997).
occurring chalcones are predominantly metabolized by Silybin (also known as silibinin or silybinin) is the main
hydroxylation (De Vincenzo et al., 2000). Among them, component of silymarin, an extract from milk thistle. Sily-
20-hydroxyl substituted chalcones have attracted much bin was able to inhibit p-nitrophenol hydroxylation via
attention, because they have biologically active properties, CYP2E1 in human liver microsomes (Zuber et al., 2002).
such as prevention of platelet aggregation (Lin et al., 1997) It displayed dose-dependent inhibition of enzyme activity
and LPS-induced septic shock (Batt et al., 1993) in animal with IC50 values in the micromolar range. However,
models. A XRE-luciferase reporter assay indicated that 20- plasma concentrations of the individual flavonolignans fol-
hydroxychalcone was most effective among five hydroxy- lowing dietary uptake do not exceed 0.5 lM(Zuber et al.,
chalcones in reducing CYP1A1 and 1B1 expression 2002), so inhibition of CYP2E1 by flavonolignans in the
through the disruption of XRE-transactivation (Wang diet may be unlikely.
et al., 2005). The inhibition on CYP1A1 was competitive
and that of CYP1B1 was non-competitive. A decrease in 2.3. CYP3A4
DMBA-DNA covalent binding was demonstrated in cul-
tures co-treated with 20-hydroxychalcone and DMBA CYP3A4 is the largest subfamily of CYP enzymes
(Wang et al., 2005). On the other hand, 2-hydroxychalcone expressed in the human liver and gastrointestinal tract. It
showed different effects; it inhibited CYP1A1 and 1B1 is involved in the metabolism of 50% of therapeutic agents
activities in recombinant microsomal preparation but as well as in the activation of toxic and carcinogenic sub-
potentiated their expression in MCF-7 cells (Wang et al., stances. It has been reported that grapefruit juice alters
2005). These findings suggested that the position and num- the pharmacokinetics of various drugs, including cyclo-
ber of hydroxyl groups in hydroxychalcone might affect the sporine (Yee et al., 1995), midazolam (Kupferschmidt
CYP1 enzyme inhibition and gene expression. et al., 1995), dihydropyridine-type calcium channel block-
A series of flavonoids isolated from a resin of the tree ers (Bailey et al., 1994), lovastatin (Kantola et al., 1998),
Dracaena cinnabari Balf. inhibited CYP1A activity in simvastatin (Lilja et al., 1998), ethinylestradiol (Weber
hepatic microsomes isolated from TCDD treated mice et al., 1996), and triazolam (Hukkinen et al., 1995). The
(Machala et al., 2001). The resin has been used in folk major mechanism for grapefruit juice-drug interactions is
198 Y.J. Moon et al. / Toxicology in Vitro 20 (2006) 187 210
thought to be due to the inhibition of intestinal CYP3A4 mice for three weeks resulted in a two-fold increase in the
by flavonoids in grapefruit juice (Ameer and Weintraub, CYP3A activity (Bray et al., 2002). The protein level of
1997; Evans, 2000; Zhang and Benet, 2001), although the CYP3A was also increased six-fold (Bray et al., 2002).
inhibition of intestinal p-glycoprotein by flavonoids has The short-term treatment may not activate the pregnane
also been observed. Particular interest has focused on the X receptor. Similarly, human studies indicated that long-
inhibitory effects of naringin (naringenin-7-rhamnogluco- term ingestion (14-days) of St. JohnÕs wort administration
side), the major flavonoid in grapefruit juice (>200 mg/ significantly induced the activity of CYP3A4 as measured
L), on the activity of intestinal CYP3A4 (Ameer and Wein- by changes in alprazolam pharmacokinetics (Markowitz
traub, 1997; Bailey et al., 2000; Evans, 2000). Naringin is et al., 2003; Wang et al., 2001), but short-term administra-
partially metabolized by gastrointestinal bacteria to form tion had no effect on CYP3A4 activity (Markowitz et al.,
the flavanone, naringenin (Bailey et al., 2000). Naringenin 2000).
exerts an inhibitory effect on intestinal CYP3A4 within Silymarin, milk thistle extract, is a naturally occurring
30 min and impairs the metabolism of the calcium channel mixture of flavonolignans (silibinin A, silibinin B, silichri-
blockers, felodipine, nitrendipine, nisoldipine, and verapa- stin, silidianin, taxifolin) (Simanek et al., 2001). It has been
mil, when co-administered with grapefruit juice (Fuhr, used to treat liver diseases for hundreds of years. It is
1998). Veronese et al. (2003) demonstrated that consump- known to protect cardiomyocytes against doxorubicin-
tion of large amounts of grapefruit juice (double strength induced oxidative stress, due mainly to its radical scaveng-
three times daily for 3 days) inhibits both intestinal and ing and iron chelating potency (Chlopcikova et al., 2004).
hepatic CYP3A4 activity, as quantified by the erythromy- Silymarin significantly decreases CYP3A4 activity (Fig. 5)
cin breath test and oral midazolam pharmacokinetics in in primary cultures of human hepatocytes (Venkatarama-
healthy male volunteers (Veronese et al., 2003). Consump- nan et al., 2000). The reason for the reduced activity of
tion of typical amounts (single strength) of grapefruit juice CYP3A4 in silymarin-treated cells is not clear at this time.
caused less inhibition of intestinal CYP3A4 (Veronese Beckmann-Knopp et al. (2000) used two model substrates,
et al., 2003). denitronifedipine and erythromycin, and showed that sily-
Another clinically important inhibitor of CYP3A4 is St. bin, the major constituent of silymarin, inhibits oxidation
JohnÕs wort (Obach, 2000). St. JohnÕs wort (Hypericum per- of denitronifedipine in a non-competitive manner, whereas
foratum) is one of the most commonly used herbal medi- the effect on erythromycin demethylation involves activa-
cines in the United States. Its major constituents include tion at low and inhibition at high silybin concentrations.
flavonols, flavonol glycosides and biflavones (Barnes CYP3A4 shows very complex behavior, in that it may have
et al., 2001; Jurgenliemk and Nahrstedt, 2002; Nahrstedt multiple conformations with distinct substrate specificities
and Butterweck, 1997). Obach (2000) using cDNA- (Koley et al., 1996). Furthermore, cooperativity in oxida-
expressed enzymes showed that hyperforin was a potent tions catalyzed by the enzyme have been described and
competitive inhibitor of CYP3A4 activities. The flavonoid attributed to the existence of more than one substrate bind-
compound I3, II8-biapigenin in St. JohnÕs wort was also ing site on the CYP3A4 molecule (Hosea et al., 2000; Ueng
shown to be a potent, competitive inhibitor of CYP3A4
activity (Obach, 2000). Quercetin in St. JohnÕs wort was
found to inhibit CYP3A4 as well (Obach, 2000; Zou
0.7
et al., 2002). However, enzyme induction with St. JohnÕs
0.6
wort has also been reported. In vitro studies using primary
cultures of human hepatocytes have demonstrated that St.
0.5
JohnÕs wort extract is a potent inducer of CYP3A4, and the
responsible component is hyperforin (Goodwin et al., 2001;
0.4
Moore et al., 2000; Wentworth et al., 2000; Zhou et al.,
0.3
2003). Hyperforin is a potent ligand (Ki of 27 nM) for the
pregnane X receptor (Moore et al., 2000), which is an
0.2
orphan nuclear receptor regulating expression of CYP3A4,
as well as other enzymes and transporters (Durr et al.,
0.1
2000; Moore et al., 2000; Wentworth et al., 2000).
*
*
Animal studies using probe drugs have provided addi-
0.0
UN RIF Sily 0.1 mM Sily 0.25 mM
tional evidence that St. JohnÕs wort is a potent modulator
Treatments
of various CYP enzymes, and the induction of CYPs
depends on the dosing regimen. Mice studies have indi- Fig. 5. Effect of silymarin on CYP3A4 activity in primary cultures of
human hepatocytes. The formation rate of 6b (OH) testosterone was
cated that short-term treatment (four consecutive days)
measured in primary cultures of human hepatocytes that were not treated
of St. JohnÕs wort extract (435 mg/kg/day) or hyperforin
or treated for 48 h with rifampin, or treated for 48 h with silymarin (0.1
(10 mg/kg/day) did not alter erythromycin N-demethylase
and 0.25 mM). , indicates significantly different from untreated cells.
*
(CYP3A) activity (Bray et al., 2002). In contrast, adminis-
Reproduced from (Venkataramanan et al., 2000), with permission from
tration of St. JohnÕs wort extract (140 or 280 mg/kg/day) to the American Society for Pharmacology and Therapteutics.
(nmol/min/mg protein)
Formation of 6-beta testosterone
Y.J. Moon et al. / Toxicology in Vitro 20 (2006) 187 210 199
et al., 1997). CYP3A4 activity, assessed by oxidation of heme were correlated with glabridin concentrations (Kent
nifedipine, was also decreased by silybin in human liver et al., 2002). Glabridin may generate reactive intermediates
microsomes (Zuber et al., 2002). The inhibition was dose- that result in heme fragmentation. Two hydroxyl groups on
dependent with IC50 values in the micromolar range. the 20 and 40 position of the flavonoid B ring of glabridin
CYP activities were unaffected by silybin at physiologically seems to be required for CYP3A4 inactivation (Kent
relevant lower concentrations (Zuber et al., 2002). Addi- et al., 2002).
tionally, milk thistle had no significant effect on CYP3A4 The number of hydroxyl groups, as well as the position
in vivo. Healthy female and male subjects have been of hydroxylation, also plays an important role in the inhib-
administered milk thistle (175 mg, twice daily, standardized itory effects of flavones. Flavones having more hydroxyl
to 80% silymarins) for 28 days with no significant changes substitutions showed stronger inhibition of CYP3A4 activ-
on phenotype ratios (1-hydroxymidazolam/midazolam) for ity than those with fewer hydroxyl groups (Ho et al., 2001).
CYP3A4 (Gurley et al., 2004). After 21 days of milk thistle In this study, the order of inhibitory potency was myrice-
extract administration (153 mg silymarin, 3 times daily), no tin > quercetin, morin > kaempferol > apigenin > flavone
clinically significant changes in the pharmacokinetics of (with 6, 5, 4, 3, 0 hydroxyl groups respectively). This was
indinavir (a CYP3A4 substrate) were noted in humans confirmed by the existence of a positive significant correla-
(Piscitelli et al., 2002). Another study of healthy volunteers tion (r = 0.89, p < 0.0005) between number of hydroxyl
also found that 20 days of silymarin ingestion (160 mg 3 groups and extent of CYP3A4 inhibition of these six com-
times daily) had no effect on the pharmacokinetics of indi- pounds. This finding is in agreement with a previous study
navir (DiCenzo et al., 2003). Administration of silymarin showing that the ability of flavonoid compounds either to
(70 mg 3 times daily) for 28 days to healthy volunteers inhibit or stimulate benzo(a)pyrene hydroxylation in
had no effect on the pharmacokinetics of aminopyrine or human liver microsomes was related to the presence or
phenylbutazone, two non-specific CYP probes (Leber and absence of hydroxyl groups respectively (Ho et al., 2001).
Knauff, 1976).
Green tea (Camellia sinensis) extract does not alter 2.4. CYP19
CYP3A4 activity in healthy volunteers (Donovan et al.,
2004). The probe drug alprazolam (2 mg, CYP3A4 activ- CYP19, also known as aromatase (11b-hydroxysteroid
ity) was administered orally to healthy volunteers dehydrogenase), is another member of the cytochrome
(n = 11) at baseline, and again after treatment with four P450 enzyme superfamily. This enzyme represents a crucial
decaffeinated green tea (DGT) capsules/day for 14 days. enzyme of estrogen biosynthesis; CYP19 converts andro-
Each DGT capsule contained 211 Ä… 25 mg of green tea cat- stenedione and testosterone to estrone and estradiol,
echins and <1 mg of caffeine. The plasma concentration of respectively (Zhu and Conney, 1998). Increased expression
the green tea flavonoid, ( )-epigallocatechin gallate of aromatase has been observed in breast cancer tissue
(EGCG), reached 1.3 Ä… 1.8 lM, 2 h after DGT treatment. (Miller et al., 1990; Zhou et al., 1996). Since estrogens
There were no significant differences in alprazolam phar- cause cell proliferation and certain estrogen metabolites
macokinetics at baseline and after DGT treatment. In this are carcinogens, local expression of CYP19 has been corre-
study, EGCG was bioavailable from this supplement lated with tumor initiation, promotion and progression.
(Donovan et al., 2004). Therefore the lack of effect of Local regulation of aromatase by both endogenous factors
DGT on CYP3A4 activity cannot be due to a lack of bio- as well as exogenous medicinal agents influences the levels
availability of the extract used in this study (Donovan of estrogen available for breast cancer growth (Brueggeme-
et al., 2004). ier et al., 2001). Flavonoids and isoflavones are structurally
Genistein and diadzein, isoflavones present in soybeans, similar to the endogenous steroid hormone, estradiol, and
were found to inhibit 3A4-mediated metabolism, but their possess either estrogenic or antiestrogenic activities (Brueg-
glycosides were inactive in human microsome preparations gemeier et al., 2001), and they are known to be competitive
(Foster et al., 2003). Genistein exhibited a similar inhibi- inhibitors of aromatase with respect to androgen sub-
tory activity against the human 3A isoform CYP3A7 (Fos- strates. Flavonoids with antiestrogenic effects have been
ter et al., 2003). The results of this study were consistent reported to be cancer preventive agents in breast cancer
with the observations of Evans, indicating that daidzein (Kumar et al., 2004).
and genistein can inhibit oxidative metabolism (Evans, Flavones have previously been reported to be competi-
2000). tive inhibitors of aromatase with respect to the androgen
Glabridin is a major flavonoid in licorice (Glycyrrhiza substrate, with Ki values at low micromolar concentrations
glabra). Kent et al. reported that the isoflavan glabridin (Adlercreutz et al., 1993; Campbell and Kurzer, 1993; Ibra-
inactivated CYP3A4 in a time-, concentration-, and him and Abul-Hajj, 1990; Kellis and Vickery, 1984; Pelis-
NADPH-dependent manner, indicative of mechanism- sero et al., 1996; Wang et al., 1994). The binding
based inactivation (Kent et al., 2002; Zhou et al., 2004). characteristics and the structural requirements necessary
Metabolism of glabridin by CYP3A4 resulted in the for the inhibition of human aromatase by flavonoids were
destruction of the heme moiety, and the loss of the obtained using computer modeling and confirmed by site-
CYP3A4-reduced carbon oxide spectrum and detectable directed mutagenesis (Kao et al., 1998). Flavonoids bind
200 Y.J. Moon et al. / Toxicology in Vitro 20 (2006) 187 210
to the active site of aromatase in an orientation in which important for both the constitutive and inducible expres-
rings A and C mimic rings D and C of the androgen sub- sion of several Phase II proteins, including QR, GST, c-
strate, respectively. Generally, flavones and flavanones glutamylcysteine synthetase and UGT. In contrast to
have higher aromatase inhibitory activity than isoflavones normal mice, nrf2 knock-out mice are more susceptible
and isoflavanones, which exhibit significant binding affini- to benzo[a]pyrene carcinogenesis and are not protected
ties for the estrogen receptor (Kao et al., 1998). Based on by Phase II inducers (Jeyapaul and Jaiswal, 2000;
the study of a series of B ring substituted flavanones with McMahon et al., 2001; Ramos-Gomez et al., 2001).
a 7-methoxy group on the A ring, the structure-activity Several papers have suggested that many of phase II
relationships suggested that hydroxylation at position 30 inducers have, or acquire by metabolism, a electrophilic
and/or 40 are the optimal pattern of B ring substitution that Michael reaction acceptor functionality (Dinkova-Kost-
enhanced the anti-aromatase activity (Pouget et al., 2002). ova, 2002; Talalay et al., 1988). Moreover, it was found
Kao et al. showed that flavones (chrysin, baicalein, and that the potency of inducers correlates with reactivity in
galangin), flavanone (naringenin) and isoflavones (biocha- the Michael reaction (Talalay et al., 1988). The inducer
nin A) inhibit the activity of human aromatase in chinese potency of Michael reaction acceptors is profoundly
hamster ovary (CHO) cells, thus decreasing estrogen bio- increased by the presence of ortho- (but not other) hydro-
synthesis and circulating estrogen levels (Kao et al., xyl substituent(s) on the aromatic ring(s) (Dinkova-Kost-
1998). Similarly, flavone, chrysin, apigenin, naringenin, ova et al., 2001). Many flavonoids contain Michael
and biochanin A inhibited human placental aromatase reaction center(s) in their molecules, thus this characteristic
(Le Bail et al., 2000). Isoflavone, equol was potent inhibitor may be related to their effects on phase II enzymes. Identi-
of the ovarian aromatase activity in rainbow trout and also fied dietary flavonoids acting as phase II inducers include:
showed inhibitory effect on human placental aromatase kaempferol (Uda et al., 1997), a flavonoid present in high
(Pelissero et al., 1996). Daily treatment for 21 days with amounts in kale; a flavonoid fraction found in blueber-
natural supplements such as propolis and honey, contain- ries/cranberries (Bomser et al., 1996); the flavolignan silib-
ing chrysin, could block the conversion of androgens into inin (also known as silybin or silybinin) obtained from milk
estrogens by inhibiting aromatase, with a consequent thistle (Zhao and Agarwal, 1999).
increase of testosterone, eventually measurable in urine
samples (Gambelunghe et al., 2003). 3.1. UDP-glucuronyltransferase
Almstrup et al. have investigated several flavonoids
using a pS2 mRNA essay for aromatase inhibition in Glucuronidation, catalyzed by the UDP-glucuronyl-
MCF-7 cells (Almstrup et al., 2002). Since aromatase con- transferase family of enzymes, is a major metabolic path-
verts testosterone to 17a-estradiol, and pS2 mRNA is reg- way of endogenous steroids, bile acids, drugs, and
ulated by estrogen, aromatase activity can be measured by carcinogens. UGTs have been divided into two families,
differences in the expression level of the pS2 mRNA after termed UGT1 and UGT2 (Mackenzie et al., 1997).
exposure to testosterone and the test compounds. Biocha- UGT1 enzymes mainly catalyse glucuronidation of exoge-
nin A, formononetin, naringenin, and chrysin are aroma- nous agents (drugs, pesticides, benzo[a]pyrene, etc.),
tase inhibitors at low concentrations (<1 lM) (Almstrup whereas UGT2 enzymes glucuronidate endogenous agents
et al., 2002). (steroid hormones and bile acids). However exceptions
exist: for instance, human UGT1A1 can metabolize the
3. Effect of bioflavonoids on phase II enzymes toxic heme breakdown product bilirubin, as well as cate-
chol estrogens, and flavonoids (King et al., 2001). In
Activation of phase II detoxifying enzymes, such as humans, glucuronidation capacity is prominently present
UDP-glucuronyl transferase (UGT), glutathione S-trans- in the liver, but UGT activity toward bile acids, phenols,
ferase (GST), and NAD(P)H:quinone oxidoreductase and bilirubin is present in human intestinal, kidney, and
(QR) by flavonoids results in the detoxification of carcino- colon tissue (Tukey and Strassburg, 2000).
gens and represents one mechanism of their anticarcino- UGT1A1, 1A3, 1A4, 1A6, and 1A9 mRNA expression
genic effects (Fig. 1). The importance of induction of is present in human livers. Of importance, UGT1A7 and
Phase II metabolism in cancer prevention has been demon- 1A10 were discovered and cloned from gastric tissue and
strated in studies of nrf-2 knockout mice; nrf-2 is a tran- UGT1A10 in biliary tissue, indicating these RNAs are
scription factor necessary for Phase II enzyme induction exclusively extrahepatic UGT1A gene products (Tukey
(Ramos-Gomez et al., 2001). Nrf2 is normally localized and Strassburg, 2000). UGT1A10 appears to be expressed
in the cytosol, where it is associated through protein-pro- in all tissues of the gastrointestinal tract except liver. This is
tein interactions with the chaperone Keap1 (Itoh et al., significant because UGT1A10 has one of the widest range
1999). The presence of an inducer disrupts the Keap1- of substrate specificities of any of the UGTs, from small
Nrf2 interactions, allowing Nrf2 to translocate to the phenolics to steroids, an indication that it may play a vital
nucleus and bind to the antioxidant/electrophile response role in most extrahepatic tissues for the glucuronidation of
element (ARE), in conjunction with small Maf proteins, endogenous and xenobiotic substrates (Tukey and Strass-
after activation (Hayes and McMahon, 2001). Nrf2 is burg, 2000). The expression of UGT2 genes also follows
Y.J. Moon et al. / Toxicology in Vitro 20 (2006) 187 210 201
a tissue-specific pattern. The human olfactory UGT2A1,
which has been shown to be one of the more versatile of
the UGTs by recognizing all of the major classes of sub-
strates, is restricted in expression to olfactory tissue. One
could predict that UGT2A1 has evolved for the need to
serve as a first line of metabolic defense for many sub-
stances that enter the body through the nasal mucosa. In
human liver, cDNAs have been identified for UGT2B4,
2B7, 2B11, and 2B15 (Tukey and Strassburg, 2000).
UGT2B7 transcripts are also found in intestine, esophagus,
brain, kidney, and pancreas. A significant observation is
that UGT2B transcripts are abundantly expressed in ste-
roid-sensitive target tissues such as prostate and mammary
gland; UGT2B10, 2B11, 2B15, and 2B17 gene transcripts
have been identified in human prostate, and UGT2B11 is
also expressed in mammary gland tissue. The presence of
UGT2B17 may have a significant impact on cancer of the
prostate gland by glucuronidating androgens and thus pro-
tecting this tissue from the carcinogenic actions of these
steroids (Tukey and Strassburg, 2000).
As reported by Walle et al., in human hepatoma HepG2
cells and the human intestinal cell line Caco-2, there was
a high level of induction of UGT1A1 by treatment with
the flavone chrysin (Walle et al., 2000). Similarly, the
UGT1A1-mediated glucuronidation of quercetin was
greatly increased (Galijatovic et al., 2001). This induction
response was quite specific because UGT1A6, UGT1A9,
and UGT2B7 were not affected by chrysin treatment.
Two of the flavonoids that induced CYP1A1, galangin Fig. 6. Induction of UGT1A1 (A) and CYP1A (B) in Hep G2 cells by
flavonoids and classical AhR inducers. Api, apigenin; Chry, chrysin; Gal,
and isorhamnetin, had no effect on the UGT1A1 activity,
galanin; Iso, isorhamnetin; , significantly higher than control (DMSO),
*
suggesting that the inducing effect of UGT1A1 is not
p < 0.001; , significantly higher than control, p < 0.05. Reproduced from
**
related to the AhR (Fig. 6) (Walle and Walle, 2002).
(Walle and Walle, 2002), with permission from the American Society for
The polymethoxyflavone tangeretin was the most potent
Pharmacology and Therapeutics.
inhibitor of UGT1A1 catalyzed estradiol-3-glucuronida-
tion in human liver microsomes (IC50 =1 lM at 5 lM
estradiol concentration) (Williams et al., 2002). Naringenin strate, long-term ingestion of green tea increases UGT
inhibited estradiol 3-glucuronidation to a similar extent at activity in rats (Bu-Abbas et al., 1995; Sohn et al., 1994),
all naringenin concentrations tested (5 100 lM) and there- and this induction is considered to contribute to the anti-
fore did not act as a competitive-type inhibitor (Williams carcinogenic effect of green tea. In contrast, three other
et al., 2002). Flavone and quercetin were weak inhibitors studies reported that quercetin has no effect on hepatic
of estradiol 3-glucuronidation at the concentrations tested NP UGT activity (Brouard et al., 1988; Canivenc-Lavier
(5 100 lM) (Williams et al., 2002). For chrysin, flavanone, et al., 1996; Siess et al., 1996). This difference may be
nobiletin, and silymarin, the greatest inhibitory effect on caused by different doses, food deprivation in animals or
estradiol 3-glucuronidation was at substrate concentrations other differences in experimental design. Since starvation
above 25 lM. is known to rapidly decrease phase II enzyme activity (Sieg-
Flavone has been shown to induce hepatic 4-nitrophenol ers et al., 1989), an initial increase in UGT enzyme activity
(NP) UGT activity in rats at a concentration of 0.25% may have disappeared during this food deprivation period.
(Brouard et al., 1988), 0.3% (Canivenc-Lavier et al., Sun et al. (1998) found that UGT was induced by
1996), and 1% (van der Logt et al., 2003) (w/w) in the diet selected flavonoids (biochanin A, diadzein, formonone-
for 2 weeks. Van der Logt et al. quantified UGT enzyme tin, genistein, prunetin, apigenin, galangin, kaempferol,
activity in hepatic and intestinal (proximal, mid and distal naringenin, and quercetin) at a concentration of 5 lMin
the prostate cancer cell line LNCaP. LNCaP cells were
small intestine and colon) tissue from male rats using NP
and 4-methlyumbelliferone as substrate (van der Logt exposed to each of the flavonoids for 6 days. Those flavo-
et al., 2003). In their study, 1% (w/w) quercetin in the basal noids stimulated the activity of testosterone-UGT, which
diet for 2 weeks caused significant increase of UGT enzyme conjugates testosterone to testosterone glucuronide. Bio-
activity in liver and proximal and distal small intestine of chanin A was the most potent inducer of UGTs (10-fold
the rats. When determined using 2-aminophenol as the sub- increase at 5 lM), with increased activity over the
202 Y.J. Moon et al. / Toxicology in Vitro 20 (2006) 187 210
concentration range of 0.5 50 lM(Sun et al., 1998). The
production and release of prostate specific antigen (PSA),
the prostatic tumor marker (Catalona et al., 1991), is tes-
tosterone dependent (Montgomery et al., 1992), and bioch-
anin A can significantly decrease PSA concentrations,
likely due to increased glucuronidation of testosterone
(Sun et al., 1998). These studies suggest that the modula-
tion of hormone metabolism by flavonoids may be impor-
tant in the prevention and treatment of prostate cancer
(Sun et al., 1998).
3.2. Glutathione-S-transferase (GST) and quinone
reductase (QR)
Flavonoids also contribute to the modulation of other
phase II detoxifying enzymes, such as GST and QR.
Human cytosolic GSTs are a family of dimeric biotransfor-
mation enzymes comprised of the four main classes; a, l, p,
and h (Hayes and Pulford, 1995). They catalyze the binding Fig. 7. Immunoblot analysis of rGSTA2 in H4IIE cells treated with
flavone. A representative immunoblot shows the level of rGSTA2 protein
of a large variety of electrophiles to the sulfydryl group of
in cells treated with PD98059 (3 30 lM) or flavone (1 10 lM) for 24 h.
glutathione, are involved in the detoxification of (oxygen)
Each lane was loaded with 10 lg of cytosolic proteins. The relative
radicals, and have a main function in the binding and
rGSTA2 level was assessed by scanning densitometry. Data represent the
transport of a wide variety of harmful compounds. GSTs
mean Ä… SD with three separate experiments (significant as compared with
* **
have a considerably important role in the detoxification control, P < 0.05; P < 0.01; control level = 1). Reproduced from (Kang
et al., 2003), with permission of Oxford University Press.
of carcinogens (Hayes and Pulford, 1995). GSTs are pres-
ent in many species and tissues and also in relatively large
amounts in the epithelial tissues of the human gastrointes-
tinal tract (Peters et al., 1991). A significant negative corre- the other hand, quercetin has been shown to effectively
inhibit human placental GST (GSTP1-1), a subclass of
lation was demonstrated between GST enzyme activity and
tumor incidence in the mucosa along the human gastroin- the GST family, in a time- and concentration-dependent
manner in vitro. GSTP1-1 activity is completely inhibited
testinal tract, suggesting the importance of GSTs in cancer
following a 1 h-incubation with 100 lM quercetin or 2 h-
prevention (Peters et al., 1993). QR prevents quinine redox
incubation with 25 lM quercetin. The inactivation mecha-
cycling and lowers levels of electrophilic quinines (Kelly
nism may involve the covalent modification of cysteine
et al., 2000). Hence, the induction of GST and QR by
flavonoids is possibly associated with cancer chemopreven- 47 in GSTP1-1 by quercetin quinone or its quinone met-
hides (van Zanden et al., 2003).
tive effects.
Genistein regulates estrogen receptor (ER)/ARE-depen- Chang et al. (1997) reported the isolation of a potent
dent gene expression in vitro (Ansell et al., 2004). ER reg- QR inducer from the pantropical coastal shrub Tephrosia
purpurea, the chalcone, (+)-tephropurpurin. Hop flavo-
ulation of a mouse GST Ya reporter gene was determined
noids (prenylchalcones and prenylflavanones) can induce
in two cell lines in the presence of 1 lM of genistein. In
GST enzymes and QR in the mouse hepatoma Hepa1c1c7
COS I cells expressing ERa and ERb, genistein repressed
cell line (Miranda et al., 2000). In contrast, the flavanone,
GST Ya ARE-dependent gene expression (Ansell et al.,
naringenin, with no prenyl group, was ineffective in induc-
2004); however, treatment of C4-12-5 cells (ER-negative
ing QR (Miranda et al., 2000). The hop chalcones, xan-
breast cancer cell line derived from the MCF-7 cell line)
with genistein resulted in modest GST gene induction fol- thohumol and dehydrocycloxanthohumol hydrate, also
induce QR activity in the AhR-defective mutant cell line,
lowing transfection with ERa and ERb. This suggests that
Hepa1c1c7bp(r)c1 (Miranda et al., 2000). Dietary admin-
the effects of genistein on GST through ER/ARE signaling
istration of morin to F344 rats led to significant increases
are cell type specific (Ansell et al., 2004).
in the activities of QR and GST in the liver, large bowel
Flavone and 20-amino-30methoxyflavone induce the
and tongue and was protective against azoxymethane-
nuclear translocation of a transcriptional factor CCAAT/
induced adenocarcinoma of the large intestine (Tanaka
enhancer-binding protein b (C/EBPb) and induce GSTA2
(a form) gene expression (Fig. 7) (Kang et al., 2003). Die- et al., 1999), as well as against 4-nitroquinone 1-oxide-
induced tongue carcinogenesis (Kawabata et al., 1999).
tary administration of flavone increases GST activities (a
and l isoforms) and the levels of glutathione in many tis- The flavonoid pinostrobin, present in honey and Thai gin-
ger (Boesenbergia pandurata) represents a potent inducer
sues of male Wistar rats (Nijhoff et al., 1995). Long-term
of mammalian QR activity (Fahey and Stephenson,
ingestion of green tea extracts (GTEs) increases cytosolic
2002). Uda et al. (1997) reported that a 2,3-double bond
GST activity in female rats (Maliakal et al., 2001). On
Y.J. Moon et al. / Toxicology in Vitro 20 (2006) 187 210 203
in the C ring of flavonoids is critical for the induction of and xenobiotic compounds (Coughtrie et al., 1998). They
QR. Among the flavonoids, the flavonols are the most are involved in the Phase II detoxification of xenobiotics,
effective inducers of QR activity in Hepa1c1c7 cells as well as in the inactivation of endogenous compounds
(kaempferol, galangin > quercetin > myricetin, apigenin such as steroid and thyroid hormones, catecholamines
(a flavone)) and flavanols and flavans are ineffective. The and bile acids (Coughtrie et al., 1998). In contrast to this
2,3-double bond in the C ring may play a role as a protective function, sulfation is also a key step in the bio-
Michael reaction acceptor (Dinkova-Kostova, 2002). In activation of a host of pro-mutagens and pro-carcinogens
contrast to flavone, flavanone, which has a saturated (Glatt, 2000; Lewis et al., 2000; Yamazoe et al., 1999). Sul-
2,3-double bond in the C ring, cannot induce QR activity fation activates carcinogens such as hydroxymethyl polycy-
(Dinkova-Kostova, 2002). clic aromatic hydrocarbons, allylic alcohols, benzylic
Silymarin can significantly elevate GST and QR activi- alcohols, and N-hydroxyarylamines, since their sulfate
ties in the liver and colon of the rat. These effects may be esters are electrophiles that covalently bind to nucleic acids
related to the in vivo suppressive effects of silymarin on and other macromolecules (Glatt, 2000; Meerman et al.,
the occurrence of aberrant crypt foci, a putative precursor 1994).
lesions for colonic adenocarcinoma, and azoxymethane- Flavonoids have been suggested as potential chemopre-
induced carcinoma (Kohno et al., 2002). ventive agents in sulfation-induced carcinogenesis (Cought-
Genistein and daidzein were found to increase QR activ- rie and Johnston, 2001; Eaton et al., 1996; Ghazali and
ities in Hepa1c1c7 cells (Yannai et al., 1998). The inhibition Waring, 1999; Tamura and Matsui, 2000; Walle et al.,
of benzo[a]pyrene metabolite-DNA binding by genistein 1995). A number of flavonoids exert inhibitory effects on
may result from QR induction (Lee et al., 1999). Induction sulfotransferase activity. A previous study has demon-
of QR activity by genistein has been also observed in the strated that flavonoids (fisetin, galangin, quercetin, myrice-
human colon cancer cell line Colo205 (Wang et al., tin, kaempferol, chrysin, apigenin and genistein) represent
1998). QR induction was further confirmed by using potent inhibitors of the P-form phenolsulfotransferase (P-
reverse transcription-polymerase chain reaction (RT- PST or SULT1A1)-mediated sulfation of acetaminophen
PCR) techniques to measure mRNA expression. A signifi- and minoxidol by human liver cytosol (Eaton et al.,
cant correlation between the expression of QR mRNA and 1996). Quercetin, fisetin, and galangin demonstrated simi-
the corresponding QR activity was observed (r = 0.76, lar potencies for the inhibition of the P-form PST while
P < 0.001) (Wang et al., 1998). The single topical applica- myricetin, chrysin, apigenin, and kaempferol were 3 10
tion of 12-O-tetradecanoyl phorbol-13-acetate (TPA), a times less potent.
well-known tumor promoter, down-regulated the level of The flavonoids daidzein, genistein, quercetin, (+)-cate-
GST activity (47%) in mouse skin model (Sharma and Sul- chin, equol and flavone are non-competitive inhibitors of
tana, 2004). Topical applications of soy isoflavones (mix- human platelet P-PST with low Km and Ki values (Ghazali
ture of 33 mg of genistein and 67 mg of daidzein), 30 min and Waring, 1999). The non-competitive nature of these
prior to the application of TPA prevented the decrease in flavonoid inhibitors of P-form PST is consistent with the
GST activity, in a dose dependent manner. Dietary geni- observation that they are poor substrates for this enzyme
stein and diadzein are also able to elevate the activities of (Table 3) (Ghazali and Waring, 1999). Consistent with pre-
GST in the kidney and QR in the colon of female rats vious findings, Harris et al. reported that quercetin was the
in vivo (Appelt and Reicks, 1999). most potent inhibitor (IC50 of 60 nM) (Harris et al., 2004).
Siess et al. (1996) showed that dietary administration of Genistein and daidzein inhibited SULT1A1 with IC50 val-
flavone to male Wistar rats elevated the activities of phase ues of 500 and 600 nM, respectively (Harris et al., 2004).
2 enzymes (GST and UGT) as well as phase I enzymes
(CYP 1A1/2 and 2B1/2) (Siess et al., 1996). Importantly,
the activities of CYP1A1/2 increased as early as 6 h after
Table 3
the first dose, reaching maximal induction after 4 days,
Km, Vmax and Ki values for inhibition of phenol sulfotransferase (PST) by
while the earliest elevation of the activities of CYP2B1/2
flavonoids
and phase II enzymes was observed 24 h after flavone feed-
Flavonoid Km, lM Vmax Ki, lM
ing. This difference in the time course of induction may
nmol/min/mg protein
suggest that the ultimate inducers of the CYP2B1/2 and
Quercetin 0.051 Ä… 0.004 0.051 Ä… 0.007 0.10 Ä… 0.03
phase II enzymes are probably flavone metabolites and
Genistein 0.047 Ä… 0.002 0.040 Ä… 0.001 0.21 Ä… 0.03
not the parent compounds themselves, or that the induc- Daidzein 0.050 Ä… 0.007 0.049 Ä… 0.004 0.34 Ä… 0.08
Equol 0.058 Ä… 0.008 0.030 Ä… 0.007 0.49 Ä… 0.11
tion occurs through different mechanisms for these different
(+)-Catechin 0.053 Ä… 0.001 0.058 Ä… 0.009 0.76 Ä… 0.03
enzymes.
Flavone 0.061 Ä… 0.004 0.031 Ä… 0.006 0.94 Ä… 0.17
Control P-form-PST activity (without inhibitor) was 0.86 Ä… 0.09 nmol/
3.3. Sulfotransferases
min/mg protein which also had a Km value of 0.050 Ä… 0.007 lM and Vmax
of 0.080 Ä… 0.03 nmol/min/mg protein. Data are expressed as means Ä… SD.
The cytosolic sulfotransferases (SULTs) catalyze the sul-
Reproduced from (Ghazali and Waring, 1999), with permission from
Elsevier Science Inc.
fate conjugation of many hormones, neurotransmitters,
204 Y.J. Moon et al. / Toxicology in Vitro 20 (2006) 187 210
The most potent inhibitor of SULT1A3 in human platelets For example, in the study of Ueng et al. (1999), naringenin
was baicalein (IC50 of 500 nM) (Harris et al., 2004). but not naringin is an inhibitor of benzo(a)pyrene hydrox-
Quercetin inhibits the sulfation of resveratrol in human ylase (AHH) activity in vitro, whereas naringin inhibited
liver and duodenum in vitro (De Santi et al., 2000). Resve- AHH activity and the expression of CYP1A2 in vivo.
ratrol is a polyphenolic compound present in wine at con- Moreover, flavonoids generally have low oral bioavailabil-
centration between 1 and 10 lM(Soleas et al., 1997). It has ity and can be degraded by gut bacteria. Therefore, concen-
beneficial effects against cancer and protective effects on the trations in vivo may not reflect the concentrations tested
cardiovascular system. The IC50 for quercetin was 12 pM under in vitro conditions (Kuhnau, 1976). Isoflavone gly-
(liver) and 15 pM (duodenum) for the inhibition of resvera- cosides are not absorbed intact across the enterocyte of
trol sulfation (De Santi et al., 2000). The potent inhibition healthy adults, and their bioavailability requires initial
of resveratrol sulfation by quercetin suggests that com- hydrolysis of the sugar moiety by intestinal b-glucosidases
pounds present in the diet may inhibit the sulphation of for uptake to the peripheral circulation (Setchell et al.,
resveratrol, thus improving its bioavailability. 2002). Apigenin, naringenin, and chrysin, which are strong
Marchetti et al. (2001) also showed that quercetin is a inhibitors of aromatase in vitro, did not inhibit androstene-
potent inhibitor of human liver and duodenum sulfotrans- dione-induced uterine growth, indicating a lack of aroma-
ferase in vitro. Drugs are often administered orally, and the tase-inhibiting effect in vivo (Saarinen et al., 2001), a
intestine and liver are therefore important sites of drug difference that may be due to their relatively poor absorp-
metabolism. In this study, cytosolic fractions of human tion and/or bioavailability. Tangeretin did not alter the
liver samples and biopsies of the duodenum were used. CYP3A4 activity in human volunteers, although it is a
The IC50 values of quercetin for the sulfation of four clin- potent stimulator of CYP3A4 activity in human liver
ically used drugs (dopamine, ( )-salbutamol, minoxidil microsomes and microsomes containing cDNA-expressed
and paracetamol) are greater in duodenum than those in CYP3A4 (Backman et al., 2000). Quercetin has been shown
liver. Such a difference may reflect the different composi- to inhibit the catalytic activity of P-PST using cell-free
tion of sulfotransferase forms in the liver and duodenum, enzyme preparations in vitro with an IC50 value as low as
since SULT1A1 is preferentially expressed in the liver 0.1 lM. In the intact human hepatoma cell line HepG2,
and SULT1A3 in the intestine (Marchetti et al., 2001). the potency of quercetin as an inhibitor of P-PST decreased
In the case of estrogen sulfotransferase (SULT1E1), about 25-fold, yielding an IC50 value of 2.5 lM(Eaton
equol was the most potent inhibitor. Equol has hydroxyl et al., 1996). This difference is possibly due to the high
groups that can potentially superimpose with the 3-hydro- serum protein binding and poor plasma membrane perme-
xyl group of 17b-estradiol (E2) (Harris et al., 2004). The ability of quercetin, and its metabolism to inactive metab-
40-hydroxyl group appears to play an important role in olites. Silymarin also has inhibitory effect on CYP3A4
the inhibitory effect because formononetin, which lacks in vitro, but not in vivo. This lack of in vitro in vivo cor-
this group, was the least potent of all the compounds relation may be due to poor bioavailability, large inter-
tested (Harris et al., 2004). Quercetin competitively inhi- individual variations in silibinin (also known as silybin or
bits the sulfation of E2 in normal human mammary epi- silybinin) absorption, lower CYP binding affinities of silib-
thelial cells (Otake et al., 2000). Another study reported inin conjugates, product variability in silymarin content, or
that quercetin, genistein and daidzein inhibit the sulfation poor dissolution characteristics of milk thistle dosage
of 17b-estradiol by zebrafish SULT (Ohkimoto et al., forms (DiCenzo et al., 2003; Gurley et al., 2004; Moore
2004). Kinetic analyses showed that the mechanism of et al., 2000).
action by these flavonoids was competitive inhibition Flavonoid metabolites may have a higher or lower bio-
(Ohkimoto et al., 2004). logical activity than the parent drug, and result in a change
The effect of the flavonoids may be mediated not just by of the overall cancer protective response. Nielsen et al.
their inhibition of the bioactivating activity of the SULTs (2000) has identified and quantified 10 different metabo-
on carcinogens, but by the effects of the sulfoconjugates lites, each bearing an intact flavan skeleton, in rat urine
of the flavonoids produced by the activity of these enzymes and feces, after repeated administration of tangeretin
(Pai et al., 2001). It has been shown that daidzein sulfocon- (abundant in citrus peal), as primarily demethylation
jugates are potent inhibitors of sterol sulfatase and and/or hydroxylation products. The differences in metabo-
decrease the production of the biologically active estro- lite formation and disposition in vitro and in vivo may
genic steroids (shown to stimulate many breast tumors) account for some of the differences observed in in vivo
in mammary tissue from their inactive sulfoconjugates and in vitro studies.
(Wong and Keung, 1997). Variable dietary exposure to a range of flavonoid com-
pounds may contribute to some of the interindividual var-
4. Difficulties in the prediction of in vivo metabolic iation in the pharmacokinetics and pharmacological
effects in humans responses observed for drugs such as phenacetin, caffeine,
and theophylline, which are substrates for CYP1A2 (Ren-
It is clear that effects of flavonoids in vivo may not dic and Di Carlo, 1997) as well as that observed for drugs
always be predicted on the basis of in vitro results alone. that are substrates for other P450 isozymes (Zhai et al.,
Y.J. Moon et al. / Toxicology in Vitro 20 (2006) 187 210 205
1998). The considerable inter-individual variability in the Acknowledgement
inhibitory effects of grapefruit juice on drug metabolism
is due in part to different bacterial strains in the gut, to We acknowledge research support through the Susan G.
CYP polymorphism, and to the differing amounts of flavo- Komen Breast Cancer Foundation, U.S. Army Breast Can-
noids present in a brand of grapefruit juice (Ameer and cer Research Program Contract DAMD17-00-1-0376 and
Weintraub, 1997; Fuhr et al., 1993). Individuals with differ- Pfizer Global Research Inc.
ent CYP profiles may derive different benefits from dietary
flavonoids with regard to protection against cancer (Brein-
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