408 HANIOKA ET AL.
¨
(Kurebayashi et al., 2002), and humans (Volkel et al.,
2002). Glucuronidation of BPA by rat liver microsomes has
been suggested to be mainly catalyzed by UDP-glucurono-
syltransferase (UGT) isoform UGT2B1 (Yokota et al.,
1999). On the other hand, Yoshihara et al. (2001, 2004)
reported that 3-hydroxy BPA (BPA catechol) and BPA o-
quinone are detected as cytochrome P450 (CYP)-dependent
metabolites in liver S9 fractions from rats, mice, monkeys,
and humans. Among these reported metabolites, BPA
monoglucuronide is completely devoid of estrogenic acti-
vity (Snyder et al., 2000; Matthews et al., 2001), and BPA
catechol and BPA o-quinone exhibit little activity (Yoshi-
hara et al., 2001). Thus, drug-metabolizing enzymes such
as CYP and UGT are closely associated with the metabo-
lism and toxicity of BPA in mammals.
MA). Yeast cell microsomes expressing human UGT1A6
(humUGT1A6) were prepared as described previously
(Hanioka et al., 2006). All other chemicals and reagents
were of the highest quality commercially available.
Assay for UGT1A6-Dependent
Glucuronidation Activities
Glucuronidation activities toward 5-HT and 4-MU were
determined as described previously (Hanioka et al., 2006)
with slight modification. The standard incubation mixture
contained 5-HT (1.25–20 mM) or 4-MU (25–400 lM) as a
substrate, microsomes from human livers or yeast cells
expressing humUGT1A6, 10 mM MgCl2, and UDPGA
(10 mM for 5-HT glucuronidation assay or 2 mM for 4-MU
glucuronidation assay) in a final volume of 500 lL of 50
mM Tris-HCl buffer (pH 7.4). Microsomal protein concen-
trations for the 5-HT glucuronidation assay were 200 lg/
mL for liver microsomes and 1000 lg/mL for yeast cell mi-
crosomes expressing UGT1A6s. Microsomal protein con-
centrations for the 4-MU glucuronidation assay were 50 lg/
mL for liver microsomes and 200 lg/mL for yeast cell mi-
crosomes expressing humUGT1A6. The substrates were
dissolved in methanol/water (20:80, v/v) for 5-HT and
methanol/dimethyl sulfoxide (50:50, v/v) for 4-MU. BPA
as an inhibitor was dissolved in methanol/dimethyl sulfox-
ide (50:50, v/v). The final concentration of the organic sol-
vent (methanol and/or dimethyl sulfoxide) in the incubation
mixture was 1% (v/v). The reaction was initiated by the
addition of UDPGA after 1-min preincubation at 378C. The
mixture was incubated for 20 min (5-HT glucuronidation
assay) or 10 min (4-MU glucuronidation assay) at 378C,
and the reaction was terminated with 50 lL of ice-cold
15% (w/v) perchloric acid. After centrifugation at 12 000
3 g for 10 min at 48C, the clear supernatant was filtered
using a 0.45-lm polytetrafluoroethylene membrane filter
(Millipore, Bedford, MA), and analyzed by high-perform-
ance liquid chromatography (HPLC) with an Inertsil ODS-
80A column (4.6 mm i.d. 3 150 mm; GL Sciences, Tokyo,
Japan). The column was maintained at 408C. 5-HT glucuro-
nide was eluted isocratically with 20 mM KH2PO4 (pH
2.5)/methanol (98:2, v/v) at a flow rate of 1.0 mL/min.
Fluorometric detection was performed with excitation at
225 nm and emission at 330 nm. 4-MU glucuronide was
eluted isocratically with 20 mM triethylamine (pH 2.1)/ace-
tonitrile (80:20, v/v) at a flow rate of 1.0 mL/min. UV
detection was performed at 318 nm.
UGTs are typical membrane proteins of the endoplasmic
reticulum and the nuclear envelope (Mackenzie et al.,
1997, 2005; Tukey and Strassburg, 2000). UGTs have been
classified into two families (UGT1 and UGT2) based on
amino acid sequence similarities (Mackenzie et al., 1997,
2005; Burchell et al., 1998). Each UGT exhibits unique
substrate and tissue specificities, and its activity or expres-
sion is influenced by genetic and environmental factors
(Mackenzie et al., 1997, 2005; Tukey and Strassburg,
2000). UGT1A6 is expressed in the liver, kidney, brain,
bile duct, stomach, and colon in humans (Burchell et al.,
1998). The expression of UGT1A6 has been shown to be
regulated by aryl hydrocarbon receptor agonists such as
2,3,7,8-tetrachlorodibenzo-p-dioxin and/or antioxidant-type
inducers such as t-butylhydroquinone in mammalian cell
lines (Bock et al., 1998, 1999); therefore, UGT1A6 is con-
sidered to be an important key enzyme in biological effects.
Although BPA is widely used in the chemical industry,
little is known about the interaction of BPA with drug-
metabolizing enzymes in humans. In this study, to clarify
the toxicity or detoxifying mechanism of BPA, the effects
of BPA on UGT1A6 activities in microsomes from livers
and yeast cells expressing UGT1A6 in humans were exam-
ined in vitro.
MATERIALS AND METHODS
Materials
BPA ([99%) was purchased from Sigma–Aldrich (St.
Louis, MO). Serotonin (5-HT) was purchased from Wako
Pure Chemical Industries (Osaka, Japan). 5-HT glucuronide
was supplied by the National Institute of Mental Health
Chemical Synthesis and Drug Supply Program (Bethesda,
MD). 4-Methylumbelliferone (4-MU), 4-MU glucuronide,
and UDP-glucuronic acid (UDPGA) were purchased from
Nacalai Tesque (Kyoto, Japan). Three human liver micro-
somes (one male, 77 years old; two females, 54 and 56
years old) were purchased from BD Biosciences (Woburn,
Inhibition Studies
UGT1A6-dependent glucuronidation activities in the ab-
sence and presence of BPA were measured according to the
method described in the previous section. For the determi-
nation of IC50 values, the substrate concentrations were set
Environmental Toxicology DOI 10.1002/tox
Hanioka, Nobumitsu
