Thioacetamide Intoxication Triggers Transcriptional Up-Regulation
but Enzyme Inactivation of UDP-Glucuronosyltransferases□
Haiping Hao, Lifang Zhang, Shan Jiang, Shiqing Sun, Ping Gong, Yuan Xie, Xueyan Zhou,
and Guangji Wang
State Key Laboratory of Natural Medicines, Key Laboratory of Drug Metabolism and Pharmacokinetics, China Pharmaceutical
University, Nanjing, People’s Republic of China
Received March 1, 2011; accepted July 6, 2011
Thioacetamide (TAA) is a potent hepatotoxicant and has been
widely used to develop experimental liver fibrosis/cirrhosis mod-
els. Although the liver toxicity of TAA has been extensively studied,
little is known about its potential influence on UDP-glucuronosyl-
transferases (UGTs) associated with the development of liver fi-
brosis. The study presented here aimed to uncover the regulation
patterns of UGTs in TAA-induced liver fibrosis of rats. Potential
counteracting effects of hepatoprotective agents were also deter-
mined. TAA treatment for 8 weeks induced a significant transcrip-
tional up-regulation of the major UGT isoforms, including UGT1A1,
UGT1A6, and UGT2B1, accompanied with the dramatic elevations
of most typical serum biomarkers of liver function and fibrosis
scores. Upon TAA intoxication, the mRNA and protein levels of the
major UGT isoforms were increased to 1.5- to 2.5-fold and 2.5- to
3.3-fold of that of the normal control, respectively. The hepatopro-
tective agents Schisandra spp. lignans extract and dimethyl diphe-
nyl bicarboxylate could largely abolish TAA-induced up-regulation
of all three UGT isoforms. However, enzyme activities of UGTs
remained unchanged after TAA treatment. The dissociation of pro-
tein expression and enzyme activity could possibly be attributed to
the inactivating effects of TAA, upon a NADPH-dependent bioac-
tivation, on UGTs. This study suggests that the transcriptional
up-regulation of UGTs may be an alternative mechanism of their
preserved activities in liver fibrosis/cirrhosis.
UDP-glucuronosyltransferases (UGTs), a group of membrane-
bound enzymes that reside in the endoplasmic reticulum, catalyze the
conjugation of UDP-glucuronic acid to the hydroxyl, carboxyl, amine,
or thiol group of numerous structurally diverse endogenous sub-
stances and xenobiotics (Radominska-Pandya et al., 2005). Because
the addition of glucuronic acids generally increases the water solu-
bility of substances and thus facilitates elimination, UGTs play an
important role in detoxifying or inactivating endogenous and xenobi-
otic substances (Wang et al., 2010). Alternatively, UGT-catalyzed
glucuronidation could contribute to the bioactivation and/or toxifica-
tion of some substances, as in the case of acyl glucuronidation of
carboxylic drugs such as nonsteroidal anti-inflammatory drugs
(Southwood et al., 2007; Koga et al., 2011). Nevertheless, the dys-
regulations of UGTs caused by various pathological factors and
exogenous toxicants could trigger significant changes of pharmaco-
logical, toxicological, and/or pathological consequences of various
The dysregulations of cytochromes P450 in hepatic fibrosis/cirrho-
sis have been extensively studied (Gomez-Lechon et al., 2009; Mor-
gan, 2009). In contrast, the current understanding of UGT dysregula-
tion in hepatic injury remains very limited, and the previous results are
largely controversial. UGT activities were found largely preserved in
liver diseases, possibly because of the activation of latent UGT
enzymes (Desmond et al., 1994), the increased expression in remain-
ing viable cells (Debinski et al., 1995), and the increased contribution
of extrahepatic UGTs (Omar et al., 1996). However, more recent
studies focusing on the mRNA levels of individual UGT isoforms
from clinical patients with liver diseases (Congiu et al., 2002) and
mice with inflammation (Richardson et al., 2006) indicated down-
regulations of most UGT isoforms. The major limitation of previous
studies concerning UGT regulation in liver injury is that the expres-
sion and enzyme activity of individual isoforms have not been con-
comitantly evaluated, resulting in inconsistencies across various stud-
ies with different measures. In addition, results obtained from clinical
patients may be complicated by other confounding factors, including
This work was supported by the National Natural Science Foundation of China
[Grants 91029746, 30801422, 30973583]; the Program for New Century Excellent
Talents in University of China [Grant NCET-09-0770]; and a Foundation for the
Author of National Excellent Doctoral Dissertation of China [Grant 200979].
H.H. and L.Z. contributed equally to this work.
S The online version of this article (available at http://dmd.aspetjournals.org)
contains supplemental material.
ABBREVIATIONS: UGTs, UDP-glucuronosyltransferases; TAA, thioacetamide; SLE, Schisandra lignans extract; DDB, dimethyl diphenyl bicar-
boxylate; TES, testosterone; 4-MU, 4-methylumbelliferone; UDPGA, UDP-glucuronic acid; HPLC, high-performance liquid chromatography; ALT,
alanine aminotransferase; AST, aspartate aminotransferase; AP, alkaline phosphatase; ALB, albumin; ?-GT, ?-glutamyl transpeptidase; T-Bil, total
bilirubin; RT-PCR, reverse transcriptase-polymerase chain reaction; PCR, polymerase chain reaction; TBST, Tris-buffered saline/Tween 20 buffer;
CMC-Na, sodium carboxymethyl cellulose; bp, base pair(s).
DRUG METABOLISM AND DISPOSITION
Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics
DMD 39:1815–1822, 2011
Vol. 39, No. 10
Printed in U.S.A.
Supplemental material to this article can be found at:
at ASPET Journals on October 29, 2015
gene polymorphisms and concomitant drugs, tobaccos, and dietary
components. Therefore, it is important to simultaneously determine
the expression and enzyme activity of individual UGT isoforms in the
experimental models of liver injury for better understanding of liver
injury per se on regulating UGTs.
Thioacetamide (TAA), originally used as a fungicide, is a potent
hepatotoxicant and has been widely used in developing experimental
liver fibrosis/cirrhosis models mimicking the human liver fibrosis/
cirrhosis (Nozu et al., 1992; Kang et al., 2005). As the largest
metabolic organ in biological systems, the liver, endowed with many
types of drug-metabolizing enzymes, plays a critical role in the
metabolic elimination of many endogenous substances and exogenous
toxicants. Therefore, extensive understanding of the dysregulation of
drug metabolizing enzymes in liver injury should be an essential step
for exploring pathological processes and consequences of liver dis-
eases. It had been previously reported that most cytochrome P450
isoforms were downregulated in a TAA-induced rat cirrhosis model
(Nakajima et al., 1998). In contrast, the profile of UGT regulation in
TAA-induced liver injury models remains largely unknown.
Considering that some recent reports suggested a dysregulation of
most individual UGT isoforms in liver injury, we hypothesized herein
that TAA-induced fibrosis/cirrhosis would also result in a dysregula-
tion of UGT isoforms. The major purpose of this study was to test the
hypothesis by concomitant evaluation of the mRNA, protein, and
enzyme activity of major rat UGT isoforms including UGT1A1, UGT
1A6, and UGT 2B1 in a TAA-induced liver injury model. In addition,
it was of great interest to determine whether hepatoprotective agents
would be effective in counteracting TAA-induced dysregulation of
UGTs. For this purpose, Schisandra spp. lignan extract (SLE) and
dimethyl diphenyl bicarboxylate (DDB), both of which had been well
proven to possess powerful hepatoprotective effects against liver
injury induced by various pathological factors(Gao et al., 2005; Ab-
del-Hameid, 2007; Xie et al., 2010), were included in this study to
further verify our hypothesis.
Materials and Methods
Chemicals and Reagents. TAA was obtained from Jiahui Medicine Chem-
ical Co. Ltd. (Anhui, China). DDB and SLE were obtained from Qingze
Science and Technology Co. Ltd. (Nanjing, China). Bicinchoninic acid protein
assay kit, SDS-polyacrylamide gel electrophoresis, and sample loading buffer
were purchased from Beyotime Institute of Biotechnology (Jiangsu, China).
UGT1A1, UGT1A6, and UGT2B antibodies (goat anti-rat IgG) were obtained
from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Bilirubin was pur-
chased from the Laboratory for the Control of Drugs of Jiangsu Province
(Nanjing, China). Testosterone (TES), 4-methylumbelliferone (4-MU), 4-MU-
O-glucuronide, glucose 6-phosphate, NADP?, glucose-6-phosphate dehydro-
genase, UDP-glucuronic acid (UDPGA), alamethicin, and D-saccharic acid
1,4-lactone monohydrate were all purchased from Sigma-Aldrich (Shanghai,
China). The purity of all of the chemicals was proven to exceed 99%.
High-performance liquid chromatography (HPLC)-grade acetonitrile and
methanol were obtained from Merck (Darmstadt, Germany). Deionized water
was purified using a Milli-Q system (Millipore Corporation, Billerica, MA).
Animals and Treatment. Male Sprague-Dawley rats (200–250 g) were
caged and fed at 20–25°C, with a 12-h light/dark cycle and free access to food
and water. The animals were acclimatized to the environment for 1 week and
fasted with free access to water for 12 h before each experiment. All animal
experimental procedures were approved by the Animal Care and Use Com-
mittee of the China Pharmaceutical University and were in accordance with the
guiding principles for the use of animals in toxicology adopted by the Chinese
Society of Toxicology. Animals were randomly divided into five groups with
six individuals in each group. Four groups of rats were injected with TAA (200
mg/kg i.p., 3% in physiological saline) twice a week for 8 consecutive weeks
to induce hepatic fibrosis (Barash et al., 2008). Another group of rats serving
as a normal control was treated with normal saline. After an initial TAA
treatment for 4 weeks, the rats were intragastrically administered with 1%
sodium carboxymethyl cellulose (CMC-Na; TAA control), SLE low dose (100
mg/kg, suspended in 1% CMC-Na), SLE high dose (400 mg/kg, in 1%
CMC-Na), or DDB (200 mg/kg, in 1% CMC-Na) once a day, respectively, for
another 4 weeks. All of the rats were weighed before treatment and at
appropriate time points throughout the whole experimental duration and were
observed daily for signs of illness. Rats were sacrificed by cervical dislocation
under ether anesthesia at the end of the 8th week. Blood samples were
collected for biochemical determinations; the liver tissues were quickly re-
moved, weighed, and frozen immediately in liquid nitrogen until analysis.
To determine the acute effects of TAA on regulating UGTs, rats were
injected with a single dose of TAA at 200 mg/kg i.p., whereas the normal
control group of rats was injected with normal saline. Six hours after treatment,
the rats were sacrificed under ether anesthesia, and the livers were quickly
removed and immediately frozen in liquid nitrogen. The liver samples were
used for the determination of mRNA levels and enzyme activities of UGTs.
Hepatic Function Biomarkers and Fibrosis Determination. Hepatic
function biomarkers, including alanine aminotransferase (ALT), aspartate ami-
notransferase (AST), alkaline phosphatase (AP), albumin (ALB), ?-glutamyl
transpeptidase (?-GT), and total bilirubin (T-Bil), were determined by an
automatic blood biochemical analyzer (Beckman counter LX20; Beckman
Coulter, Inc., Fullerton, CA).
Slices of the same part of each rat liver were cut off and fixed in phosphate-
buffered 10% formaldehyde solution and then embedded in paraffin wax.
Sections of liver tissues with 5-?m thickness were cut and stained with
hematoxylin-eosin according to standard procedures and then examined for
histopathological changes under the microscope (Olympus BH2; Olympus,
Tokyo, Japan). The extent of fibrosis was graded, according to the semiquan-
titative METAVIR scoring system, as no fibrosis (score 0), portal fibrosis
without septa (score 1), portal fibrosis with few septa (score 2), numerous septa
without cirrhosis (score 3), and established cirrhosis (score 4). The assessment of
the extent of fibrosis was performed in blind by an experienced pathologist.
RT-PCR Analysis. Frozen liver samples were crushed with a glass mortar
in liquid nitrogen. RNA was extracted using a RNA extraction kit (Keygene,
Shanghai, China) according to the manufacturer’s instructions. The RNA pellet
was dried and dissolved in RNase-free water and stored at ?80°C until use.
RNA concentrations were determined with a spectrophotometer at 260 nm. For
cDNA synthesis, a reaction media containing 5 ?g of total RNA, 1 ?l of oligo
dT (10 ?M), 1 ?l of dNTPs (10 mM), and RNase-free water to a final volume
of 15 ?l was heated at 65°C for 5 min and then immediately placed in an
ice-water bath for 5 min. To each sample, 0.5 ?l of RNase inhibitor (40 U), 2
?l of 10? avian myeloblastosis virus reaction buffer, 1 ?l of dithiothreitol (1
M), and 1 ?l of reverse transcriptase (avian myeloblastosis virus) was added
and kept at 37°C for 60 min and then at 70°C for 15 min. The cDNA samples
were kept at ?80°C until use.
Primers for rat UGT1A1, UGT1A6, UGT2B1, and glyceraldehyde phos-
phate dehydrogenase (GADPH) were designed using the primer select soft-
ware (Primer Premier 5.0; PREMIER Biosoft International, Palo Alto, CA) on
the basis of previous reports (Nozu et al., 1992; Kang et al., 2005). Sequences
of the polymerase chain reaction (PCR) primers are as follows (sense and
antisense for each isoform, 5?–3?, respectively): UGT1A1 (554 bp), ACG AAG
TGG TGG TCA TAG CA, CTG TAA GAT TTC AGT GGC AAG; UGT1A6
(270 bp), TGG TGC TAG TGC CAG AAG TCA A, GAG CAT CAA ACT
GGT TCT CCC T; UGT2B1 (439 bp), GTC ACG GTT CTT GTA TCT TCG
G, GAA CAA CAG GCA CAT AGG AAG G; and GAPDH (352 bp), CGG
GAA GCT TGT CAT CAA TGG, GGC AGT GAT GGC ATG GAC TG. The
PCR mixture containing 5 ?l of 10? Taq buffer, 1 ?l of dNTPs (10 mM), 2
?l of each primer (10 ?M), 2 ?l of cDNA template, 0.5 ?l of Taq DNA
polymerase, and 4 ?l of MgCl2(2.5 mM) was initially denatured at 95°C for
90 s, followed by 32 cycles of amplification for UGT1A1, 30 cycles for
UGT1A6, 28 cycles for UGT2B1, or 30 cycles for GAPDH. Each cycle was
performed as the following steps: 90 s at 94°C (denaturation); 45 s at 59.5°C
for UGT1A1, 59°C for UGT1A6, 56°C for UGT2B1, or 55°C for GAPDH
(annealing); and 45 s at 72°C (extension). The final extension was performed
at 72°C for 10 min. Then, 8 ?l of the PCR-amplified mixture was subjected to
electrophoresis on 1.5% agarose gel and visualized by ethidium bromide
staining. The gel was then photographed by UV gel image formation (Gel
HAO ET AL.
at ASPET Journals on October 29, 2015
DocTM XR; Bio-Rad Laboratories, Hercules, CA), and the gray scale was
determined by a digitalized software (Quantity one; Bio-Rad Laboratories).
Western Immunoblotting. Liver tissue proteins (70 ?g) were resolved by
SDS-10% polyacrylamide gel electrophoresis and transferred to a polyvi-
nylidene difluoride membrane at 180 mA for 60 min using a wet transfer
method (Bio-Rad Laboratories, wet blotter). After transferring, the membranes
were blocked with 5% nonfat dry milk in Tris-buffered saline/Tween 20 buffer
(TBST) at 37°C for 90 min. The membranes were then incubated overnight at
4°C with each diluted antibody (1:200 for UGT1A1, 1:150 for UGT1A6, and
1:200 for UGT2B) in 5% nonfat dry milk-TBST and washed 3 times with
TBST for 15 min each time. For references, ?-actin was detected using a
polyclonal antibody (1:200). A peroxidase-conjugated rabbit anti-goat IgG
(BioWorld Products, Inc., Visalia, CA) (1:5000) was used as second antibody
and incubated with the membranes for 60 min at 37°C. The membranes were
washed extensively again (three times, 15 min/time) with TBST before detec-
tion using enhanced chemiluminescence detection reagents (BioWorld). After
exposure in the darkroom, the film was then photographed and the signal
intensity was detected by a digitalized software (Quantity one, Bio-Rad). The
protein levels of UGTs were normalized to that of the reference band ?-actin.
Preparation of Rat Liver Microsomes. For enzyme activities analysis of
UGTs, each group of rats’ livers were pooled together and the hepatic micro-
somal suspensions were prepared by differential centrifugation as described
previously (Hao et al., 2007) and resuspended in 0.25 M sucrose/1 mM EDTA
solution, pH 7.4. Protein concentrations were determined with a bicinchoninic
acid protein assay kit according to the manufacturer’s instructions. Final
protein concentration was adjusted to approximately 10 mg/ml with the same
buffer. The suspensions were frozen and maintained at ?80°C until use.
Enzyme Activities Assay of UGTs. Enzyme activities of UGTs in rat liver
microsomes were determined using the typical substrate bilirubin for
UGT1A1, 4-MU for UGT1A6, and TES for UGT2B1. For the UGT1A1 test,
the pooled and alamethicin-permeabilized microsomes (2 mg/ml) from each
group were incubated for 30 min at 37°C in the presence of 10 mM MgCl2and
320 ?M bilirubin in a final volume of 0.2 ml. The reactions were initiated by
the addition of 20 mM UDPGA and terminated by the addition of 200 ?l of
ice-cold glycine/HCl (2 M, pH 2.7). The bilirubin glucuronidation was assayed
by the electrophotometrical determination of metabolites as described else-
where with minor modifications (Gordon et al., 1983). Bilirubin glucuronides
were determined by measuring the absorbance at 550 nm using a detection kit
on the basis of the conversion of mono- and di-bilirubin glucuronides to
dipyrrolic azo derivatives. Because bilirubin is a light-sensitive compound, all
handling procedures for bilirubin were performed under dim light.
For the UGT1A6 and UGT2B1 tests, the pooled and alamethicin-
permeabilized microsomes (2 mg/ml) of each group were incubated at 37°C
with 4-MU (500 ?M) for 5 min or TES (75 ?M) for 18 min in the media
containing 10 mM MgCl2in a final volume of 0.2 ml in phosphate-buffered
saline, pH 7.4. The reactions were initiated by the addition of 20 mM UDPGA
and terminated by the addition of 200 ?l of ice-cold acetonitrile. After
centrifugation, 10 ?l of supernatant was injected into a HPLC system (Shi-
madzu, Kyoto, Japan) for the detection of 4-MU, TES, and their glucuronides.
The UV detector was set at 220 nm for the detection of 4-MU and its
glucuronide and at 250 nm for TES and its glucuronide, respectively. Sepa-
ration was achieved on a Diamonsil C18 column (150 ? 4.6 mm, 5 ?m; Dikma
Technologies, Inc., Lake Forest, CA). A mobile phase consisting acetonitrile
and 50 mM ammonium phosphate buffer [30:70 (v/v)] was used for TES and
its glucuronide detection. For 4-MU and its glucuronide detection, the mobile
phase was composed of acetonitrile and 20 mM ammonium phosphate buffer
[10:90 (v/v)]. The flow rate was set at 1.0 ml/min in both cases.
To determine the potential inactivating effects of TAA on UGTs, TAA (5,
50, and 200 ?M) was preincubated with rat liver microsomes for 0.5 h with or
without the addition of NADPH before the addition of UDPGA and probe
substrates to initiate the UGT-catalyzed reactions. Effects of hydrogen perox-
ide (0.2, 1, and 5 mM) preincubation were also determined likewise. In another
set of incubations, DDB (10, 50, and 200 ?M) or GSH (1 and 5 mM) was
added concomitantly with TAA in the UGT1A6 activity test.
TAA Disposition in Liver Microsomes. To investigate whether the hepa-
toprotective agents were influential on the TAA disposition, an assay of
NADPH-dependent metabolic depletion of TAA was performed in the in vitro
liver microsome incubation system. The incubation mixture contained 1 mg/ml
microsome protein, TAA (0.1 mM), NADPH-regenerating system, and the
hepatoprotective agent SLE (0–500 ?g/ml) or its lignan component schizan-
drol A (0–200 ?M) and deoxyschizandrin (0–100 ?M), or DDB (0–500 ?M),
in a final volume of 200 ?l. NADPH-regeneration system was finally added to
initiate the reaction. All of the incubations were conducted at 37°C for 0.5 h
and terminated by adding ice-cold acetonitrile. The concentration of remaining
TAA was determined by HPLC-UV (Chilakapati et al., 2005). Five replicates
were performed for each of the incubations.
Statistical Analysis. Data are expressed as mean ? S.D., and statistically
significant differences were assessed with the one-way analysis of variance
followed by post hoc analysis (Dunnett’s test) in most cases, except for the
liver fibrosis scores, which were analyzed by a ?2test. All statistical analyses
were performed with SPSS software version 16.0 (SPSS Inc., Chicago, IL); the
difference was considered statistically significant when the probability value
was less than 0.05 (P ? 0.05).
Body and Liver Weight. TAA treatment for 8 weeks caused a
significant growth retardation of rats, as evidenced by the significant
decrease of final body weight and body weight gains. Although the
absolute liver weights were not changed significantly, TAA treatment
for 8 weeks induced a significant increase of liver weight index. SLE
or DDB treatment exerted a slight ameliorating effect on the body
weight loss, but little impact on the increase of liver weight index,
induced by TAA (Supplemental Table 1).
Histopathology. Induction of hepatic fibrosis was notable in rats
exposed to TAA for 8 weeks, characterized with a fibrosis score of
2.83 ? 0.41. SLE and DDB exerted a significant attenuating effect on
TAA-induced hepatic fibrosis, as evidenced from histopathological
examinations (Supplemental Fig. 1) and fibrosis scores (Table 1).
Serum ALT, AST, AP, Albumin, ?-GT, and Total Bilirubin.
Typical serum biomarkers of hepatic functions including ALT,
AST, AP, ALB, ?-GT, and T-Bil were determined. The results are
shown in Fig. 1. Compared with the normal control, the levels of
all serum biomarkers except ALB were significantly increased
after TAA treatment for 8 weeks, indicating a serious hepatic
injury caused by TAA. Rats treated with SLE and DDB showed
remarkably lower levels of serum biomarkers compared with those
in the TAA group.
mRNA Levels of UGTs. The mRNA levels for the major rat liver
UGTs including UGT1A1, UGT1A6, and UGT2B1 were determined
by RT-PCR. TAA treatment significantly increased the mRNA levels
of all three UGT isoforms in rat livers, with UGT1A1 increased to
182%, UGT1A6 to 157%, and UGT2B1 to 206% of those in the
control group (Fig. 2). It was of interest to find that treatment with the
hepatoprotective agents SLE and DDB exhibited a significant coun-
teracting effect on the TAA-induced adaptive mRNA up-regulation of
UGTs. The mRNA levels of all three UGT isoforms in the SLE (400
mg/kg) and DDB treatment groups were even restored to the normal
levels (97–132% of the normal control).
Liver fibrosis scores of rats with TAA intoxication for 8 weeks
The extent of fibrosis was graded according to the semiquantitative METAVIR scoring
system and scored as 0, 1, 2, 3, and 4, respectively. The liver fibrosis for rats of normal
control was graded as zero.
Fibrosis Grading Scores (Number of Rats)
Mean ? S.D.
2.83 ? 0.41
2.17 ? 0.75
1.83 ? 0.98*
1.83 ? 0.75*
* P ? 0.05 compared with TAA group.
TAA-INDUCED DYSREGULATIONS OF UGT
at ASPET Journals on October 29, 2015
Protein Expression of UGTs. For assessing the protein expression
levels of the major rat liver UGTs, a Western blotting analysis using
the polyclonal antibody of UGT1A1, UGT1A6, and UGT2B was
applied in this study. In accordance with the mRNA levels, TAA
treatment resulted in a nearly 3-fold increase of the protein levels of
all three UGT isoforms compared with the normal control (Fig. 3).
SLE and DDB treatment significantly counteracted the TAA-induced
up-regulation of UGT protein levels. High-dose SLE (400 mg/kg) and
DDB treatment restored the UGT protein levels back to normal levels
(97–135% of normal control).
To evaluate the regulating effects of SLE and DDB per se on UGTs,
SLE (400 mg/kg) or DDB (200 mg/kg) was administered intragastri-
cally to healthy rats once a day for 2 weeks, and the protein levels of
hepatic UGTs were determined by western blotting. As a result, no
significant differences were found between the hepatoprotective-
agent-treated groups and the control group, suggesting that SLE and
DDB themselves have little direct effect on regulating UGTs (Sup-
plemental Fig. 2).
Enzyme Activities of UGTs. Enzyme activities for the major rat UGT
isoforms UGT1A1, UGT1A6, and UGT2B1 were determined using the
typical substrate bilirubin, 4-MU, and TES, respectively, in pooled liver
microsome incubation systems. The incubation conditions including micro-
somal protein levels and incubation time were optimized to ensure a linear
production of glucuronidation metabolites of each substrate.
In contrast to the UGT mRNA and protein expressions, it was
surprising to find that TAA treatment exhibited little effect on the
enzyme activities of all three UGT isoforms tested in the microsomal
incubation systems. In addition, neither SLE nor DDB treatment
showed significant influence on the enzyme activities of UGTs com-
pared with either the normal control or TAA group (Fig. 4).
The lack of correlation between protein expression and enzyme
activity upon TAA intoxication prompted us to hypothesize that TAA
may directly inactivate UGTs. To test this hypothesis, TAA (0, 5, 50,
and 200 ?M) was preincubated in liver microsomes with the addition
of NADPH for 0.5 h, and then the remaining enzyme activities of
UGTs were determined by adding the respective probe substrates as
described under Enzyme Activities Assay of UGTs. The preincubation
of TAA without the addition of NADPH showed little effect on UGT
activities. In contrast, TAA preincubation with NADPH dramatically
and concentration-dependently inactivated the enzyme activities of all
three UGT isoforms (Fig. 5). Likewise, hydrogen peroxide pretreat-
ment significantly inactivated the enzyme activities of UGTs (Fig. 6).
Taking the UGT1A6 test as an example, the addition of DDB and GSH
partially abolished TAA-induced inactivation of enzyme activity (Fig. 7).
Acute Effects of TAA on Regulating UGTs in Rats. To observe
the acute effects of TAA on regulating UGTs, rats were intraperito-
FIG. 2. RT-PCR analysis of the mRNA levels of UGT1A1, UGT1A6, and
UGT2B1. a, image of RT-PCR results; b, semiquantitative data analysis of RT-PCR
results. The mRNA levels of UGTs were normalized to that of GADPH in the same
preparation and quantitated by densitometry. Data are expressed as percentage (%)
of control, and bars represent mean ? S.D. of six individual liver samples. ??, P ?
0.01 compared with the control group; ‚‚, P ? 0.01 compared with TAA group.
FIG. 1. Biochemical parameters of liver function after 8 weeks of TAA intoxica-
tions. The serum level of T-Bil, ALT, AST, AP, ?-GT, and ALB for the control
group is 2.96 ? 0.25 ?M, 44.40 ? 3.21 IU/l, 149.25 ? 18.01 IU/l, 191.80 ? 10.66
U/l, 1.20 ? 0.45 U/l, and 19.80 ? 0.84 g/l, respectively. Data are expressed as
percentage (%) of control (mean ? S.D., n ? 6). ?, P ? 0.05 and ??, P ? 0.01
compared with the control group; ‚, P ? 0.05 and ‚‚, P ? 0.01 compared with
FIG. 3. Immunoblot analysis of the protein levels of UGT1A1, UGT1A6, and
UGT2B1. a, image of Western blot results; b, semiquantitative data analysis of
Western blot results. The protein levels of UGTs were normalized to that of ?-actin
in the same preparation and quantitated by densitometry. Each of the UGTs protein
blots is a representative of six liver samples. Densitometry data are expressed as
percentage (%) of the control, and bars represent mean ? S.D. of six liver samples.
??, P ? 0.01 compared with the control group; ‚, P ? 0.05 and ‚‚, P ? 0.01
compared with TAA group.
HAO ET AL.
at ASPET Journals on October 29, 2015
neally injected with a single dose of TAA (200 mg/kg). The mRNA
levels and enzyme activities of liver microsomal UGTs after a 6-h
TAA treatment were determined. As shown in Fig. 8, a single dose of
TAA treatment exerted little influence on the mRNA levels and
enzyme activities of most UGTs, except for a slight reduction of
Effect of DDB and SLE on TAA Disposition In Vitro. To
determine whether SLE and DDB could influence TAA disposition,
the NADPH-dependent metabolic depletion of TAA was determined
in microsomal incubation systems in vitro. The influences of DDB
and schizandrol A on the metabolic elimination of TAA were very
limited. SLE and its lignan component deoxyschizandrin at high
concentrations exhibited slight inhibitory effects on the metabolic
elimination of TAA in microsomes; however, such a high concentra-
tion would be difficult to achieve at in vivo conditions (Fig. 9).
As a typical hepatotoxicant, TAA induces a progressive liver fi-
brosis and cirrhosis in experimental animals that mimics the patho-
logical processes and consequences of liver cirrhosis in human beings.
The study presented here contributes to disclose that the mRNA and
protein expression levels of the major UGT isoforms in rats, including
UGT1A1, UGT1A6, and UGT2B1, are adaptively upregulated in a
TAA-induced rat liver fibrosis model, whereas the enzyme activities
of UGTs remain largely unchanged. The dissociation of protein ex-
pression and enzyme activity is potentially attributed to the inactivat-
ing effects of TAA upon NADPH-dependent bioactivation on UGTs.
The hepatoprotective agents SLE and DDB elicit a significant coun-
teracting effect on the adaptive change of UGT expressions, accom-
panied with powerful hepatoprotective effects as evidenced from the
examinations of serum biomarkers and histopathology.
In accordance with previous reports (Karantonis et al., 2010;
Shaker et al., 2010), the study presented here confirmed that TAA
treatment resulted in a significant retardation of natural body growth,
liver hypertrophy, elevated serum biomarkers of liver function, and
the progressive liver fibrosis. As expected, SLE and DDB treatments
exhibited powerful hepatoprotective effects, supported by the signif-
icant recovery of most indexes determined. The hepatoprotective
effect of SLE (and its lignans components), a well known herbal
medicine not only widely used in China but also used as a component
of Kampo medicines and dietary supplements (Mu et al., 2006), had
been previously proven in many other experimental models of liver
injury. DDB, an intermediate produced from the chemical synthesis of
schisandrin C, had been clinically used for more than 30 years in the
treatment of viral and chemical hepatitis (Gao and Kang, 2006;
Abdel-Hameid, 2007). The model-independent hepatoprotective ef-
fects of SLE and DDB may be explained by their powerful antioxi-
dative effects (Chiu et al., 2003; Ko and Chiu, 2005).
Although the pathological processes and underlying mechanisms of
TAA-induced liver fibrosis/cirrhosis have been extensively studied,
little is known about the potential dysregulation of drug metabolizing
enzymes in this model. The study presented here found that the major
rat UGT isoforms, including UGT1A1, UGT1A6, and UGT2B1, were
transcriptionally upregulated after an 8-consecutive-week TAA intox-
ication. Likewise, the protein levels of all three isoforms were in-
creased in parallel with the mRNA up-regulation. The references in
the current literature concerning the transcriptional regulation of
UGTs in liver injury are largely controversial. Some reports showed
that most UGT isoforms were transcriptionally downregulated in the
liver samples from humans with liver diseases, which was found to
correlate with inflammation but not with fibrosis scores (Congiu et al.,
2002), and from the liver samples of mice treated with lipopolysac-
charides or bacterial infections (Richardson et al., 2006). However,
another set of reports witnessed an up-regulation of most UGT iso-
forms in humans with cirrhosis (Debinski et al., 1995) and rat liver
samples (Debinski et al., 1996), as well as in regenerated rat liver
tissues after partial hepatectomy (Pellizzer et al., 1996). Such discrep-
ancies across different researches may be explained by the diverse
severity and property of liver diseases, various pathological factors,
and different animal models applied. The large individual variability
of UGT isoforms in human beings caused by potential genetic and
environmental factors and the exposures to drugs and dietary compo-
nents further complicated the determination of liver injury per se on
FIG. 5. The inactivating effects of TAA on the enzyme activities of UGT1A1,
UGT1A6, and UGT2B1. TAA was preincubated in liver microsomes with the
addition of NADPH regenerating system for 0.5 h before the enzyme activity assay
of UGTs. The preincubation of TAA without the addition of NADPH had little
effect on UGT activities. Data are expressed as percentage (%) of control, and bars
represent mean ? S.D. of five incubations. ??, P ? 0.01 compared with the control
FIG. 6. The inactivating effects of hydrogen peroxide on UGT1A1, UGT1A6, and
UGT2B1. Hydrogen peroxide (200 ?M, 1 mM, and 5 mM) was preincubated with
microsomes for 0.5 h before the enzyme activity test of UGTs. Data are expressed
as percentage (%) of control; bars represent mean ? S.D. of five incubations. ??,
P ? 0.01 compared with the control group.
FIG. 4. Enzyme activities of UGT1A1, UGT1A6, and UGT2B1. Bilirubin (320
?M), 4-MU (500 ?M), or TES (75 ?M) was used as the typical substrate for
UGT1A1, UGT1A6, and UGT2B1, respectively. Data are expressed as percentage
(%) of control; bars represent mean ? S.D. of five replicates.
TAA-INDUCED DYSREGULATIONS OF UGT
at ASPET Journals on October 29, 2015
It is important to note that most previous studies concerning UGT
regulations in liver injury have not performed a concomitant assay of
mRNA, protein, and enzyme activity, which may constitute another
cause for the controversial results obtained in different researches. We
found that despite a 2- to 3-fold up-regulation of mRNA and protein
levels after 8 weeks of TAA treatment, the enzyme activities of all
three UGT isoforms remain unchanged. A similar phenomenon had
been found in a previous study, in which the influence of octachlo-
rostyrene treatment on inducing UGT mRNA levels was much more
prominent than that on the glucuronidation activity toward 1-naphthol
(Yanagiba et al., 2009). It is now well known that TAA is bioactivated
mainly by CYP2E1 to produce reactive intermediates and the metab-
olite di-S-oxide, which is highly reactive against macromolecules by
covalent binding and/or oxidative modifications (Kang et al., 2008).
We hypothesized that the enzyme activity of UGTs may be inacti-
vated by the reactive metabolites and/or reactive oxygen species
generated from the process of TAA bioactivation. To test this hypoth-
esis, microsomes obtained from healthy rats were preincubated with
TAA or hydrogen peroxide for 0.5 h, and then the remaining enzyme
activities of UGTs were determined. The results confirmed that the
enzyme activity of all three UGT isoforms was dramatically inacti-
vated by TAA treatment, providing a good explanation of TAA-
induced unparallel change between the expressions and activities of
UGTs. It is important to note that the inactivating effect of TAA on
the enzyme activities of UGTs is NADPH-dependent, suggesting that
the metabolic bioactivation is necessary for TAA to inactivate UGTs.
FIG. 7. The protective effects of DDB and GSH against the inac-
tivating effect of TAA on UGT1A6. NADPH regenerating system
was included in all groups of samples except the TAA 200 ?M (?)
group. Data are expressed as mean ? S.D. of five incubations. ??,
P ? 0.01 compared with the TAA 200 ?M (?) group; ‚‚, P ?
0.01 compared with the TAA 200 ?M (?) group.
FIG. 8. Acute effects of TAA on regulating mRNA and enzyme activities of UGTs
in rats. a, mRNA levels of UGT1A1, UGT1A6, and UGT2B1. The mRNA levels of
UGTs were normalized to that of GADPH in the same preparation and quantitated
by densitometry. b, enzyme activities of UGTs. Data are expressed as percentage
(%) of control; bars represent mean ? S.D. of triplicate samples. ?, P ? 0.05
compared with the control group.
FIG. 9. Effects of DDB and SLE and its major components on the metabolic
depletion of TAA in microsomal incubation systems. a, effects of DDB and SLE; b,
effects of deoxyschizandrin and schizandrol A. TAA (0.1 mM) was incubated in
liver microsome incubation systems containing NADPH regenerating system for
0.5 h at 37°C. Velocity of TAA metabolic depletion was calculated by measuring
the loss of TAA in the incubation media. Data are expressed as mean ? S.D. of five
incubations. ?, P ? 0.05 compared with control incubations (without the addition of
HAO ET AL.
at ASPET Journals on October 29, 2015
Hydrogen peroxide can also inactivate the enzyme activities of UGTs;
however, a relatively high concentration is necessary to take effect.
This evidence suggests that the reactive metabolites produced from
TAA bioactivation may play an important role in inactivating UGTs.
However, the contribution of TAA-induced oxidative stress cannot be
excluded, considering that GSH and DDB pretreatment partially abol-
ished TAA-induced inactivation of UGTs. Because DDB has little
effect on the metabolic depletion of TAA in liver microsomes (Fig. 9),
its effect on protecting against the TAA inactivating effect on UGTs
is unlikely to have resulted from its influence on TAA bioactivation.
Further research is necessary to determine whether such effects of
DDB and GSH are attributed to their antioxidant activities and/or
capacities of trapping TAA reactive metabolites.
The potential influences of single-dose treatment of TAA to rats
were further evaluated to determine whether the long-term TAA-
intoxication-induced transcriptional up-regulation of UGTs was asso-
ciated with liver fibrosis progression or merely an acute effect of
TAA. The results showed that single-dose treatment of TAA had little
effect on regulating the mRNA levels and enzyme activities of UGTs.
This seems controversial between the in vivo and in vitro assay in
terms of the inactivating effect of TAA on UGTs. However, it would
be understandable considering that some in vivo protective machinery
such as antioxidant and the trapping of reactive metabolites might
prevent the enzyme from inactivation by a single dose of TAA
treatment. Together, these results suggest that the transcriptional
up-regulation of UGTs after long-term TAA treatment is more likely
an adaptive response from the progression of liver fibrosis, and that
repeated doses of TAA intoxication may be necessary to inactivate
UGTs in vivo. The major limitation of this study is that the underlying
molecular mechanisms of TAA-induced up-regulation of UGTs have
not been addressed. TAA-induced liver injury was typically charac-
terized with oxidative stress and lipid peroxidation (Low et al., 2004;
Natarajan et al., 2006; Aller et al., 2008), and the antioxidant treat-
ment was very effective in preventing TAA-induced liver injury
(Hyoudou et al., 2007; Tsai et al., 2010). In addition, we found in the
study presented here that SLE and DDB, both proven as good anti-
oxidants, could significantly attenuate TAA-induced liver injury and
transcriptional up-regulation of UGTs. These lines of evidence sug-
gest that TAA-triggered oxidative stress might be an underlying factor
contributing to the transcriptional up-regulation of UGTs. Therefore,
it will be of interest to determine in future studies whether nuclear
factor-like-2, an oxidative stress-responsive transcription factor, is
involved in TAA-induced up-regulation of UGTs considering that
some reports documented an important role of nuclear factor-like-2 on
regulating UGTs (Enomoto et al., 2001; Umemura et al., 2006;
Yeager et al., 2009).
In conclusion, TAA-induced liver fibrosis leads to a transcriptional
up-regulation of UGTs; however, the enzyme activities of UGTs
remain largely unchanged. The dissociation between enzyme expres-
sion and activity is possibly attributed to the UGT inactivating effects
of TAA upon bioactivation. SLE and DDB can largely abolish TAA-
induced transcriptional up-regulation of UGTs despite the hepatopro-
tective agents themselves possessing little effect on regulating UGTs.
Results obtained from the study presented here may provide novel
explanations on the preserved UGT activities in liver injury and may
shed a light on understanding the influence of hepatoprotective agents
on regulating UGTs in liver injury.
Participated in research design: Hao and Wang.
Conducted experiments: Hao, Zhang, Jiang, Sun, Xie, and Zhou.
Performed data analysis: Hao, Zhang, Jiang, and Gong.
Wrote or contributed to the writing of the manuscript: Hao, Zhang, Jiang,
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Address correspondence to: Guangji Wang, State Key Laboratory of Natural
Medicines, Key Laboratory of Drug Metabolism and Pharmacokinetics, China Pharma-
ceutical University, 24 Tongjiaxiang, Nanjing, China. E-mail: guangjiwang@hotmail.
HAO ET AL.
at ASPET Journals on October 29, 2015