Differential UGT1A1 Induction by Chrysin in Primary Human
Hepatocytes and HepG2 Cells
Cornelia M. Smith, Richard A. Graham, Wojciech L. Krol, Ivin S. Silver, Masahiko Negishi,
Hongbing Wang, and Edward L. Lecluyse1
Division of Drug Delivery and Disposition, School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North
Carolina (C.M.S., R.A.G., H.W., E.L.L.); GlaxoSmithKline, Research Triangle Park, North Carolina (R.A.G., W.L.K., I.S.S.); and
National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina (M.N.)
Received June 9, 2005; accepted August 30, 2005
Chrysin, a dietary flavonoid, has been shown to markedly in-
duce UGT1A1 expression and activity in HepG2 and Caco-2
cell lines; thus, it has been suggested to have clinical utility in
the treatment of UGT1A1-mediated deficiencies, such as un-
conjugated hyperbilirubinemia or the prevention of 7-ethyl-10-
hydroxycamptothecin (SN-38) toxicity. However, little is known
about its induction potential in a more physiologically relevant
model system, such as primary hepatocyte culture. In this
study, induction of UGT1A1 expression (mRNA, protein, and
activity) was investigated in primary human hepatocyte cultures
after treatment with chrysin and other prototypical inducers.
Endogenous nuclear receptor-mediated UGT1A1 induction
was studied using transient transfection reporter assays in
primary human hepatocytes and HepG2 cells. Results indi-
cated that induction of UGT1A1 expression was minimal in
human hepatocytes treated with chrysin compared with that in
HepG2 cells (1.2- versus 11-fold, respectively). Subsequent
experiments to determine whether the differential response was
due to its metabolic stability revealed strikingly different elimi-
nation rate constants between the two cell systems (half-life of
13 min in human hepatocytes versus 122 min in HepG2 cell
suspensions). Further study demonstrated that UGT1A1 mRNA
expression could be induced in human hepatocyte cultures by
either increasing the chrysin dosing frequency or by modulating
chrysin metabolism, suggesting that the differential induction
observed in hepatocytes and HepG2 cells was due to differ-
ences in the metabolic clearance of chrysin. In conclusion, this
study suggests that the metabolic stability of chrysin likely
would limit its ability to induce UGT1A1 in vivo.
The potential use of flavonoids as beneficial and therapeu-
tic agents has gained widespread popularity over the years.
The bioflavonoid chrysin, in particular, has been assessed by
various investigators and proposed to be advantageous as an
antioxidant, anticancer, anxiolytic, and anti-human immu-
nodeficiency virus agent (Critchfield et al., 1996; Paladini et
al., 1999; Chan et al., 2000; Galijatovic et al., 2001). Chrysin
is naturally present in small amounts in honey, vegetables,
fruits, and beverages (Uhl et al., 2003). Additionally, chrysin
is sold as an androgen-boosting supplement due to its mod-
ulation of aromatase activity (Kellis and Vickery, 1984; Kao
et al., 1998).
The mechanism(s) by which chrysin exerts these pharma-
cological effects has been explored, particularly its antioxi-
dant and anticancer effects. These beneficial effects were
deemed to result from the modulation of drug-metabolizing
enzymes and those involved in the regulation of cellular
oxidation processes. Through various in vivo and in vitro
experiments, primarily conducted in rodents, chrysin was
shown to induce or inhibit CYP1A activities (Tsyrlov et al.,
1994; Moon et al., 1998; Breinholt et al., 1999), and chrysin
metabolism was investigated in rat liver microsomes
(Nielsen et al., 1998).
This study was supported by National Institutes of Health Grant DK061652
and a National Institutes of Health fellowship (to C.M.S.). This work was
completed at the University of North Carolina at Chapel Hill where E.L.L. was
previously employed as an associate professor.
1Current affiliation: CellzDirect, Pittsboro, North Carolina.
Article, publication date, and citation information can be found at
ABBREVIATIONS: UGT, uridine diphosphate glucuronosyltransferase; HH, primary human hepatocyte(s); CAR, constitutive androstane receptor;
PXR, pregnane X receptor; AhR, aryl hydrocarbon receptor; 3-MC, 3-methylcholanthrene; PB, phenobarbital; DMSO, dimethyl sulfoxide; E2,
estradiol; E3G, estradiol-3-glucuronide; BORN, borneol; HPLC, high-performance liquid chromatography; DMEM, Dulbecco’s modified Eagle’s
medium; ITS?, insulin, transferrin, selenium, linoleic acid, and bovine serum albumin; CTL, control; gtPBREM, UGT1A1 phenobarbital-responsive
enhancer module; PBREM, phenobarbital-responsive enhancer module; XREM, xenobiotic responsive enhancer module; PCR, polyacrylamide gel
electrophoresis; AhRE, aryl hydrocarbon receptor response element; NR, nuclear receptor; CHRY, chrysin; Gluc, glucuronide; Sulf, sulfate; MULTI,
multiple dosing; STD, standard dosing.
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
U.S. Government work not protected by U.S. copyright
JPET 315:1256–1264, 2005
Vol. 315, No. 3
Printed in U.S.A.
at ASPET Journals on December 31, 2015
Because studies in humans were limited, other investiga-
tors examined disposition, metabolism, and induction poten-
tial of chrysin in clinical studies or immortalized human cell
lines. The bioavailability of chrysin seemed to be very poor,
and maximal plasma concentrations (Cmax) were low (12–64
nM) after the administration of a single 400-mg oral dose in
human volunteers (Walle et al., 2001). Furthermore, chrysin
was found to be metabolized by UDP-glucuronosyltrans-
ferases (UGTs) and sulfotransferases in humans, Caco-2, and
HepG2 cells (Galijatovic et al., 1999; Walle et al., 2001). More
than one isoform belonging to the UGT1A subfamily was
involved in the glucuronidation of chrysin. Chrysin was
shown to be a substrate of UGT1A1, UGT1A6, and UGT1A9
with Vmaxvalues of 360, 157, and 1210 pmol/min/mg protein,
respectively (Walle et al., 2000).
Additionally, chrysin was identified as an inducer of UGT
in vitro because glucuronidation was greatly increased (14-
fold over untreated) as determined by Vmax/Kmin the chry-
sin-treated HepG2 cell homogenate (Walle et al., 2000). The
increased in vitro clearance was due specifically to induction
of UGT1A1 because chrysin did not induce UGT1A6,
UGT1A9, or UGT1A4 (Walle et al., 2000). Moreover,
UGT1A1-mediated bilirubin glucuronidation was increased
20-fold in HepG2 cells after treatment with 25 ?M chrysin
(Walle et al., 2000). These studies established that a phase II
biotransformation reaction was the predominant clearance
mechanism for the elimination of chrysin and depicted that
chrysin might possess a great potential to induce UGT1A1 in
Typically, the UGT enzymes have not been found to be
induced to the same extent as cytochrome P450 enzymes in
parallel studies. Three- to 4-fold induction of UGT1A1, at
best, has been observed in our laboratory and those of others
using prototypical inducing agents in primary human hepa-
tocytes (HH) (Sutherland et al., 1993; Brierley et al., 1996;
Ritter et al., 1999; Soars et al., 2004; Smith et al., 2005). As
such, the large UGT1A1 induction response observed in im-
mortalized cell lines after chrysin exposure seems rather
atypical. Immortalized cell lines may not be the best model
system for assessment of enzyme induction, because impor-
tant transcription factors, cofactors, transporters, and some
drug-metabolizing enzymes may be decreased or absent com-
pared with other more physiologically relevant models.
As part of this study, a more systematic evaluation of the
induction of UGT1A1 by chrysin was performed using HH,
which are more representative of in vivo conditions (Le-
Cluyse, 2001). Because the expression of drug-metabolizing
enzymes, transporters, and transcriptional factors is greater
in HH than in immortalized hepatoma cell lines, it was
hypothesized that chrysin would not induce UGT1A1 expres-
sion significantly in HH due to extensive metabolism. Exper-
iments were conducted in both HH and HepG2 cell cultures
to determine the induction of UGT1A1 mRNA, immunoreac-
tive protein, and activity after treatment with chrysin over a
range of concentrations. Because the UGT1A1 gene contains
functional elements that are responsive to constitutive an-
drostane receptor (CAR), pregnane X receptor (PXR), and
aryl hydrocarbon receptor (AhR) agonists (Sugatani et al.,
2001; Xie et al., 2003; Yueh et al., 2003), transient transfec-
tion assays were conducted in both HH and HepG2 cells to
better understand the mechanism of its regulation by chry-
sin. Metabolic stability studies were undertaken in suspen-
sions of freshly isolated and cryopreserved human hepato-
cytes and HepG2 cells to determine the differences in chrysin
clearance. Borneol, an inhibitor of glucuronidation (Watkins
and Klaassen, 1983), was used to aid further the character-
ization of the cell-dependent chrysin induction response.
Materials and Methods
Chemicals. Chrysin (CHRY), 3-methylcholanthrene (3-MC), phe-
nobarbital (PB), dimethyl sulfoxide (DMSO), perchloric acid, ?-es-
tradiol (E2), ?-estradiol-3-glucuronide (E3G), dexamethasone, dex-
tromethorphan, and borneol (BORN) were purchased from Sigma-
Aldrich (St. Louis, MO). HPLC grade acetonitrile, formic acid, and
potassium phosphate were purchased from Fisher Scientific Co.
(Pittsburgh, PA). Casein solution (10?) was obtained from Vector
Laboratories (Burlingame, CA). All other chemicals were of HPLC or
highest grade available commercially.
Biological Reagents. Dulbecco’s modified Eagle’s medium
(DMEM), and modified Chee’s medium were purchased from Invitro-
gen (Carlsbad, CA). Rat-tail collagen Matrigel and insulin, trans-
ferrin, selenium, linoleic acid, and bovine serum albumin (ITS?)
were obtained from BD Biosciences Discovery Labware (Bedford,
MA). The human hepatoma cell line HepG2 was purchased from
American Type Culture Collection (Manassas, VA). Collagenase type
IV was from Sigma-Aldrich. TRIzol reagent was from Invitrogen. A
polyclonal antibody against human UGT1A1 was purchased from BD
Biosciences Discovery Labware, and CYP1A2 antibody was from
Chemicon International (Temecula, CA). Horseradish peroxidase-
conjugated goat anti-rabbit IgG antibody was from Zymed Labora-
tories (South San Francisco, CA). CellPhect transfection kit was
purchased from GE Healthcare (Little Chalfont, Buckinghamshire,
UK). The dual-luciferase reporter assay System was from Promega
(Madison, WI). All other biological reagents were purchased from
commercial suppliers, and they were either of American Chemical
Society or molecular biology grade.
Isolation and Culturing of Human Hepatocytes. Human
hepatocytes were obtained either by isolation from liver tissues ob-
tained from University of North Carolina Hospitals or from com-
mercial sources (CellzDirect, Tucson, AZ; ADMET Technologies,
Research Triangle Park, NC). Hepatocyte isolations performed in the
investigators’ laboratory were conducted as described by LeCluyse et
al. (2005). Hepatocytes were cultured on Biocoat plates (BD Bio-
sciences Discovery Labware) and overlaid with Matrigel (LeCluyse
et al., 2005). Characteristics of human liver donors are provided in
Table 1, where “L” represents the human liver number.
Induction Studies. Cultures of hepatocytes were maintained in
modified Chee’s medium supplemented with ITS?and 0.1 ?M dexa-
methasone for 36 to 48 h before exposure to inducers. HepG2 cells
were cultured in DMEM supplemented with 10% fetal bovine serum
and penicillin-streptomycin (Invitrogen). Hepatocytes or HepG2 cells
were treated with vehicle (0.1% DMSO) alone, chrysin (1–50 ?M), or
3-MC (1 or 5 ?M) for three consecutive days for protein expression or
catalytic activity assessment. Levels of UGT1A1 mRNA were as-
sessed following a 24- to 72-h treatment period. In a separate study,
HH were exposed to either a single dose of 10 ?M chrysin or repeat
doses of 10 ?M chrysin every 2 h for 12 h. Control (CTL) groups
included a single dose of 0.1% DMSO and multiple dosing of 0.1%
DMSO every 2 h for 12 h. At the end of each treatment period, cells
were harvested in TRIzol reagent or homogenization buffer (50 mM
Tris-HCl, pH 7.4, 150 mM KCl, and 2 mM EDTA) (LeCluyse et al.,
2005). Protein concentrations were determined following the manu-
facturer’s instructions using a commercially available kit (BCA pro-
tein assay; Pierce Chemical, Rockford, IL).
Plasmid Constructs. A 290-base pair DNA (?3483/?3194) frag-
ment containing the human UGT1A1 phenobarbital-responsive en-
hancer module (gtPBREM) was cloned into a pGL3TK-firefly lucif-
erase reporter plasmid as described previously (Sugatani et al.,
Induction of UGT1A1 by Chrysin in Human Hepatocytes
at ASPET Journals on December 31, 2015
latently infected cells by flavonoid compounds. AIDS Res Hum Retroviruses 12:
Galijatovic A, Otake Y, Walle UK, and Walle T (1999) Extensive metabolism of the
flavonoid chrysin by human Caco-2 and Hep G2 cells. Xenobiotica 29:1241–1256.
Galijatovic A, Otake Y, Walle UK, and Walle T (2001) Induction of UDP-
glucuronosyltransferase UGT1A1 by the flavonoid chrysin in Caco-2 cells–
potential role in carcinogen bioinactivation. Pharm Res (NY) 18:374–379.
Galijatovic A, Walle UK, and Walle T (2000) Induction of UDP-glucuronosyltrans-
ferase by the flavonoids chrysin and quercetin in Caco-2 cells. Pharm Res (NY)
Grillo MP, Hua F, Knutson CG, Ware JA, and Li C (2003) Mechanistic studies on the
bioactivation of diclofenac: identification of diclofenac-S-acyl-glutathione in vitro
in incubations with rat and human hepatocytes. Chem Res Toxicol 16:1410–1417.
Kao YC, Zhou C, Sherman M, Laughton CA, and Chen S (1998) Molecular basis of
the inhibition of human aromatase (estrogen synthetase) by flavone and isoflavone
phytoestrogens: a site-directed mutagenesis study. Environ Health Perspect 106:
Kellis JT Jr and Vickery LE (1984) Inhibition of human estrogen synthetase (aro-
matase) by flavones. Science (Wash DC) 225:1032–1034.
Kretz-Rommel A and Boelsterli UA (1993) Diclofenac covalent protein binding is
dependent on acyl glucuronide formation and is inversely related to P450-
mediated acute cell injury in cultured rat hepatocytes. Toxicol Appl Pharmacol
LeCluyse EL (2001) Human hepatocyte culture systems for the in vitro evaluation of
cytochrome P450 expression and regulation. Eur J Pharm Sci 13:343–368.
LeCluyse EL, Alexandre E, Hamilton GA, Viollon-Abadie C, Coon DJ, Jolley S, and
Richert L (2005) Isolation and culture of primary human hepatocytes. Methods
Mol Biol 290:207–229.
Lemaire G, de Sousa G, and Rahmani R (2004) A PXR reporter gene assay in a stable
cell culture system: CYP3A4 and CYP2B6 induction by pesticides. Biochem Phar-
Li W, Harper PA, Tang BK, and Okey AB (1998) Regulation of cytochrome P450
enzymes by aryl hydrocarbon receptor in human cells: CYP1A2 expression in the
LS180 colon carcinoma cell line after treatment with 2,3,7,8-tetrachlorodibenzo-
p-dioxin or 3-methylcholanthrene. Biochem Pharmacol 56:599–612.
Moon JY, Lee DW, and Park KH (1998) Inhibition of 7-ethoxycoumarin O-deethylase
activity in rat liver microsomes by naturally occurring flavonoids: structure-
activity relationships. Xenobiotica 28:117–126.
Nielsen SE, Breinholt V, Justesen U, Cornett C, and Dragsted LO (1998) In vitro
biotransformation of flavonoids by rat liver microsomes. Xenobiotica 28:389–401.
Paladini AC, Marder M, Viola H, Wolfman C, Wasowski C, and Medina JH (1999)
Flavonoids and the central nervous system: from forgotten factors to potent anxi-
olytic compounds. J Pharm Pharmacol 51:519–526.
Porubek DJ, Grillo MP, and Baillie TA (1989) The covalent binding to protein of
valproic acid and its hepatotoxic metabolite, 2-n-propyl-4-pentenoic acid, in rats
and in isolated rat hepatocytes. Drug Metab Dispos 17:123–130.
Rencurel F, Stenhouse A, Hawley SA, Friedberg T, Hardie DG, Sutherland C, and
Wolf CR (2005) AMP-activated protein kinase mediates phenobarbital induction of
CYP2B gene expression in hepatocytes and a newly derived human hepatoma cell
line. J Biol Chem 280:4367–4373.
Ritter JK, Kessler FK, Thompson MT, Grove AD, Auyeung DJ, and Fisher RA (1999)
Expression and inducibility of the human bilirubin UDP-glucuronosyltransferase
UGT1A1 in liver and cultured primary hepatocytes: evidence for both genetic and
environmental influences. Hepatology 30:476–484.
Roberts EA, Johnson KC, Harper PA, and Okey AB (1990) Characterization of the Ah
receptor mediating aryl hydrocarbon hydroxylase induction in the human liver cell
line Hep G2. Arch Biochem Biophys 276:442–450.
Smith CM, Faucette SR, Wang H, and Lecluyse EL (2005) Modulation of UDP-
glucuronosyltransferase 1A1 in primary human hepatocytes by prototypical in-
ducers. J Biochem Mol Toxicol 19:96–108.
Soars MG, Petullo DM, Eckstein JA, Kasper SC, and Wrighton SA (2004) An
assessment of UDP-glucuronosyltransferase induction using primary human
hepatocytes. Drug Metab Dispos 32:140–148.
Sueyoshi T, Kawamoto T, Zelko I, Honkakoski P, and Negishi M (1999) The re-
pressed nuclear receptor CAR responds to phenobarbital in activating the human
CYP2B6 gene. J Biol Chem 274:6043–6046.
Sugatani J, Kojima H, Ueda A, Kakizaki S, Yoshinari K, Gong QH, Owens IS,
Negishi M, and Sueyoshi T (2001) The phenobarbital response enhancer module in
the human bilirubin UDP-glucuronosyltransferase UGT1A1 gene and regulation
by the nuclear receptor CAR. Hepatology 33:1232–1238.
Sugatani J, Yamakawa K, Tonda E, Nishitani S, Yoshinari K, Degawa M, Abe I,
Noguchi H, and Miwa M (2004) The induction of human UDP-glucuronosyltrans-
ferase 1A1 mediated through a distal enhancer module by flavonoids and xenobi-
otics. Biochem Pharmacol 67:989–1000.
Sutherland L, Ebner T, and Burchell B (1993) The expression of UDP-glucuronosyl-
transferases of the UGT1 family in human liver and kidney and in response to
drugs. Biochem Pharmacol 45:295–301.
Tsyrlov IB, Mikhailenko VM, and Gelboin HV (1994) Isozyme- and species-specific
susceptibility of cDNA-expressed CYP1A P-450s to different flavonoids. Biochim
Biophys Acta 1205:325–335.
Uhl M, Ecker S, Kassie F, Lhoste E, Chakraborty A, Mohn G, and Knasmuller S
(2003) Effect of chrysin, a flavonoid compound, on the mutagenic activity of
2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and benzo(a)pyrene
(B(a)P) in bacterial and human hepatoma (HepG2) cells. Arch Toxicol 77:477–484.
Walle T, Otake Y, Brubaker JA, Walle UK, and Halushka PV (2001) Disposition and
metabolism of the flavonoid chrysin in normal volunteers. Br J Clin Pharmacol
Walle T, Otake Y, Galijatovic A, Ritter JK, and Walle UK (2000) Induction of
UDP-glucuronosyltransferase UGT1A1 by the flavonoid chrysin in the human
hepatoma cell line Hep G2. Drug Metab Dispos 28:1077–1082.
Walle UK and Walle T (2002) Induction of human UDP-glucuronosyltransferase
UGT1A1 by flavonoids-structural requirements. Drug Metab Dispos 30:564–569.
Wang H, Faucette S, Sueyoshi T, Moore R, Ferguson S, Negishi M, and LeCluyse EL
(2003) A novel distal enhancer module regulated by pregnane X receptor/
constitutive androstane receptor is essential for the maximal induction of CYP2B6
gene expression. J Biol Chem 278:14146–14152.
Wang LQ, Falany CN, and James MO (2004) Triclosan as a substrate and inhibitor
of 3?-phosphoadenosine 5?-phosphosulfate-sulfotransferase and UDP-glucuronosyl
transferase in human liver fractions. Drug Metab Dispos 32:1162–1169.
Watkins JB and Klaassen CD (1983) Chemically-induced alteration of UDP-
glucuronic acid concentration in rat liver. Drug Metab Dispos 11:37–40.
Xie W, Yeuh MF, Radominska-Pandya A, Saini SP, Negishi Y, Bottroff BS, Cabrera
GY, Tukey RH, and Evans RM (2003) Control of steroid, heme and carcinogen
metabolism by nuclear pregnane X receptor and constitutive androstane receptor.
Proc Natl Acad Sci USA 100:4150–4155.
Yueh MF, Huang YH, Hiller A, Chen S, Nguyen N, and Tukey RH (2003) Involve-
ment of the xenobiotic response element (XRE) in Ah receptor-mediated induction
of human UDP-glucuronosyltransferase 1A1. J Biol Chem 278:15001–15006.
Zhang S, Qin C, and Safe SH (2003) Flavonoids as aryl hydrocarbon receptor
agonists/antagonists: effects of structure and cell context. Environ Health Perspect
Address correspondence to: Dr. Hongbing Wang, Division of Drug Delivery
and Disposition, School of Pharmacy, CB 7360, Kerr Hall, Room 2319, Uni-
versity of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7360. E-mail:
Smith et al.
at ASPET Journals on December 31, 2015