Transcription Factor-Mediated Regulation of ...
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Transcription Factor-Mediated Regulation of Carboxylesterase Enzymes in Livers
of Mice
Youcai Zhang, Xingguo Cheng, Lauren Aleksunes, and Curtis D. Klaassen
Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas
Medical Center, 3901 Rainbow Boulevard, Kansas City, KS, 66160, USA (Y.Z., X.C.,
L.A., and C.D.K)
DMD Fast Forward. Published on March 19, 2012 as doi:10.1124/dmd.111.043877
Copyright 2012 by the American Society for Pharmacology and Experimental Therapeutics.
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Running title: Chemical regulation of mouse carboxylesterases
Corresponding to: Curtis D. Klaassen, Ph.D.
Department of Pharmacology, Toxicology, and Therapeutics
University of Kansas Medical Center
3901 Rainbow Boulevard
Kansas City, KS 66160
Phone: 1-913-588-7714
Fax: 1-913-588-7501
E-mail: [email protected]
Text pages: 35
Tables: 1
Figures: 7
References: 51
Words in the Abstract: 243
Words in the Introduction: 667
Words in the Discussion: 1480
Abbreviations: AhR, aryl hydrocarbon receptor; bDNA, branched DNA signal amplification assay; BHA, butylated hydroxyanisole; BNF, β-naphthoflavone; CAR, constitutive androstane receptor; CES, carboxylesterase; CLOF, clofibric acid; CPFB, ciprofibrate; DAS, diallyl sulfide; DEHP, diethylhexylphthalate; DEX, dexamethasone; ETH, ethoxyquin; Gapdh, glyceraldehydes-3-phosphate dehydrogenase; GR, glucocorticoid receptor; HNF4α, hepatocyte nuclear factor-4-alpha; IL, interleukin; MEI, microsomal enzyme inducer; Nqo1, NAD(P)H:quinone oxidoreductase 1; Nrf2, nuclear factor erythroid 2-related factor 2; OPZ, oltipraz; PB, phenobarbital; PCB126, polychlorinated biphenyl 126; PCN, pregnenolone-16α-carbonitrile; PXR, pregnane X receptor; PPARα, peroxisome proliferator activated receptor alpha; RLUs, relative light
units; SPR, spironolactone; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; TCPOBOP, 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene; WT, wild-type.
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Abstract
The induction of drug-metabolizing enzymes by chemicals is one of the major reasons
for drug-drug interactions. In the present study, the regulation of the mRNA expression
of one arylacetamide deacetylase (Aadac) gene and eleven carboxylesterase (Ces)
genes by fifteen microsomal enzyme inducers (MEIs) was examined in livers of male
C57BL/6 mice. The data demonstrated that mRNA of Aadac is suppressed by three aryl
hydrocarbon receptor (AhR) ligands, two constitutive androstane receptor (CAR)
activators, two pregnane X receptor (PXR) ligands, and one nuclear factor erythroid 2-
related factor 2 (Nrf2) activator. The mRNA of Ces1 subfamily is not altered by most of
the MEIs, whereas the mRNA of Ces2 subfamily is readily induced by the activators of
CAR, PXR, and Nrf2, but not peroxisome proliferator activated receptor alpha (PPARα).
Studies using null mice demonstrated that: 1) AhR is required for the 2,3,7,8-
tetrachlorodibenzo-p-dioxin (TCDD)-mediated suppression of Aadac and Ces3a; 2)
CAR is involved in the 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP)-mediated
induction of Aadac, Ces2c, 2a, and 3a; 3) PXR is required for the pregnenolone-16α-
carbonitrile (PCN)-mediated induction of Aadac, Ces2c, and 2a; 4) Nrf2 is required for
the oltipraz (OPZ)-mediated induction of Ces1g and 2c; and 5) PXR is not required for
the DEX-mediated suppression of Cess in livers of mice. In conclusion, the present
study systematically investigated the regulation of Cess by MEIs in livers of mice, and
demonstrated that MEIs modulate mRNA expression of mouse hepatic Cess via the
activation of AhR, CAR, PXR, and/or Nrf2 transcriptional pathways.
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Introduction
Carboxylesterases (rodents: Cess; human:CESs) catalyze the hydrolysis of many
ester- and amide-containing chemicals, as well as drugs and prodrugs, to their
respective free acids (Satoh and Hosokawa, 1998). Based upon the extent of amino
acid homology and substrate selectivity, Satoh and Hosokawa classified the CES/Ces
enzymes across species into five groups, and revealed that the majority of identified
CES/Ces enzymes belong to either the CES1 or CES2 subfamily (Satoh and Hosokawa,
1998; Satoh and Hosokawa, 2006). Arylacetamide deacetylase (rodents: Aadac;
human: AADAC) is an important enzyme in the metabolic activation of arylamine
substrates to ultimate carcinogens, and also functions as a microsomal lipase during
lipoprotein secretion (Probst et al., 1994; Trickett et al., 2001). AADAC/Aadac was
categorized into CES5 family, with a structure different from other four CES families
(Satoh and Hosokawa, 2006; Hosokawa et al., 2008).
Due to the confusion in naming rodent Ces genes, a new nomenclature system
has been recommended for the five mammalian carboxylesterase gene families in
human, mouse, and rat (Holmes et al., 2010). In the new nomenclature system, six
human CESs on human chromosome 16, fifteen rat Cess on rat chromosome 19, and
twenty mouse Cess on mouse chromosome 8 have been recognized and described
(Holmes et al., 2010). However, AADAC/Aadac genes were not included in this new
system, probably due to their different chromosomal location than other CES/Ces genes.
CESs/Cess are found in a number of tissues including liver, kidney, small intestine,
heart, lung, brain, testis, nasal, and respiratory tissues, adipose tissues, leukocytes, and
blood (Satoh and Hosokawa, 1998). Among various tissues, the highest hydrolase
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activity is present in liver. Microsomal enzyme inducers (MEIs) exert their effects on
target genes through the direct or indirect activation of transcription factors, such as aryl
hydrocarbon receptor (AhR), pregnane X receptor (PXR), constitutive androstane
receptor (CAR), peroxisome proliferator activated receptor alpha (PPARα), and nuclear
factor erythroid 2-related factor 2 (Nrf2) (Handschin and Meyer, 2003; Numazawa and
Yoshida, 2004). CES/Ces activity has been previously shown to be induced
phenobarbital (PB), aroclor1254, polycyclic aromatic hydrocarbons, synthetic
glucocorticoids, pregnenolone-16α-carbonitrile (PCN), and clofibrate (CLOF) (Satoh and
Hosokawa, 1998). CAR and PXR have been shown to play important roles in the
regulation of mouse Cess (Staudinger et al., 2010). Potential transcriptional factors that
depict binding sites in CES/Ces promoters include specificity protein 1 (Sp1), Sp3,
CCAAT/enhancer binding protein (C/EBP), upstream stimulatory factor 1 (USF1),
neurofibromin 1(NF-1), nuclear factor kappa B (NFkB), PPARα, glucocorticoid receptor
(GR), sterol regulatory element binding protein (SREBP), hepatocyte nuclear factor 1
(HNF1), HNF3, and HNF4 binding sites (Satoh and Hosokawa, 2006).
CES enzymes are considered to be one of the major determinants of the
metabolism and disposition of ester-containing drugs through their actions in liver and
intestine. CES substrates include numerous clinically prescribed anticancer prodrugs,
carbamate and pyrethroid insecticides, as well as environmental toxicants and
procarcinogens (Hosokawa et al., 2008). Moreover, an ester linkage methodology is
frequently utilized in rational drug design in order to target a prodrug, or to improve the
water solubility of a novel compound. Thus, it is of clinical significance to investigate the
regulation of CESs/Cess. Previous studies focused mainly on the regulation of human
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and rat CESs/Cess. However, information on the regulation of mouse Cess is limited.
Therefore, the purpose of the present study was to systematically investigate the
regulation of Cess in livers of mice by prototypical MEIs that respectively activate 5
transcription factors. The present study focused on 1 mouse Aadac gene and 11
mouse Ces genes, which have been previously investigated, including Ces1c
(previously called Es1) (Genetta et al., 1988), Ces1d (previously Ces3) (Dolinsky et al.,
2001), Ces1e (previously Es22 or egasyn) (Ovnic et al., 1991), Ces1f (previously Es4 or
TGH-2) (Poole et al., 2001), Ces1g (previously Ces1 or Est1) (Ellinghaus et al., 1998),
Ces2a (previously Ces6) (The MGC Project Team 2004), Ces2b (previous Ces2A7)
(Satoh and Hosokawa, 2006), Ces2c (previously Ces2) (Furihata et al., 2003), Ces2e
(previously Ces5) (The MGC Project Team 2004), Ces3a (previously esterase 31 or
Est31)(Aida et al., 1993), and Ces5a (previously Ces7) (Miyazaki et al., 2003).
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Materials and Methods
Chemicals. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) was a kind gift from Dr.
Karl Rozman (University of Kansas Medical Center, Kansas City, KS). Oltipraz (OPZ)
was purchased from LKT laboratories, Inc. (St. Paul, MN). Polychlorinated biphenyl
126 (PCB126) was obtained from AccuStandard (New Haven, CT). β-Naphthoflavone
(BNF), diallyl sulfide (DAS), clofibric acid (CLOF), di-(2-ethylhexyl)-phthalate,
ethoxyquin (EXQ), dexamethasone (DEX), pregnenolone-16α-carbonitrile (PCN),
ciprofibrate, butylated hydroxyanisole (BHA), spironolactone (SPR), phenobarbital (PB),
and 1,4-bis[2-(3,5-dichloropuridyloxy)]benzene (TCPOBOP) were purchased from
Sigma-Aldrich Co. (St. Louis, MO). Sodium chloride, HEPES sodium salt, HEPES free
acid, lithium lauryl sulfate, EDTA, and D-(+)-glucose were purchased from Sigma-Aldrich
(St. Louis, MO). Micro-O-protect was purchased from Roche Diagnostics (Indianapolis,
IN). Chloroform, agarose, and ethidium bromide were purchased from AMRESCO Inc.
(Solon, OH). RNA Bee was purchased from TelTest Inc. (Friendswood, TX). All other
chemicals, unless indicated, were purchased from Sigma-Aldrich Co. (St. Louis, MO).
Animals. Eight-week old male C57BL/6 mice were purchased from Jackson
Laboratories (Bar Harbor, Maine). Male AhR-null mice (>99% congenic for C57BL/6J
background) from Jackson laboratories (stock #002831) have been described
previously (Schmidt et al., 1996). Breeding pairs of CAR-null mice in the C57BL/6
background were obtained as described previously (Ueda A, 2002). Breeding pairs of
PXR-null mice (Staudinger JL, 2001) in the C57BL/6 background were kindly provided
by Dr. Frank J. Gonzalez (National Institutes of Health/National Cancer Institute,
Bethesda, MD). PPARα-null mice originally engineered by the laboratory of Dr. Frank J.
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Gonzalez (Lee SS, 1995) were backcrossed to a C57BL/6 background (Akiyama TE,
2001). Breeding pairs of Nrf2-null mice on a mixed C57BL/6 and AKR background were
provided by Dr. Jefferson Chan (University of California, Irvine) (Chan et al., 1996) and
backcrossed to a C57BL/6 background in our animal care facility. All mice were housed
in an American Animal Associations Laboratory Animal Care (AALAC) accredited facility
with a 12:12 hr light:dark cycle and provided chow and water ad libitum.
Microsomal Enzyme Inducer Treatment. Adult ( approximately 8-weeks of age)
male C57BL/6 mice were treated with five groups of prototypical drug-metabolizing
enzyme inducers as previously described (Cheng et al., 2005). Each class contained
three chemicals that are known to activate a specific signaling pathway. The dose and
dosing regimen of these compounds were previously shown to induce the
corresponding target genes in mice, namely Cyp1A1 for AhR ligands, Cyp2B1 for CAR
activators, Cyp3A11 for PXR ligands, Cyp4A14 for PPARα ligands, and
NAD(P)H:quinine oxidoreductase 1 (Nqo1) for Nrf2 activators (Cheng et al., 2005).
Similar inductions were also observed in the present study (Supplemental Fig 1).
In another experiment, five knockout mouse models and their corresponding wild-
type mice (n=5/group) were treated with one of five prototypical MEIs once daily for 4
days: AhR-null mice (TCDD, 40 µg/kg, ip in corn oil); CAR-null mice
(TCPOBOP, 300 μg/kg, ip in corn oil); PXR-null mice (PCN, 200 mg/kg, ip in corn oil);
PPARα-null mice (CLFB, 500 mg/kg, ip in saline); and Nrf2-null mice (OPZ, 150 mg/kg,
po in corn oil). Livers were collected on day 5, frozen in liquid nitrogen, and stored at -
80°C.
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Low-dose and High-dose Dexamethasone Treatment. DEX at higher doses
(20-50mg/kg) have been shown to activate PXR in vivo (Xie et al., 2000), whereas lower
doses of DEX (2-6mg/kg) have little effect on PXR activation (Heuman et al., 1982). To
investigate the mechanism of DEX in regulation of mouse Cess, both a low dose (2
mg/kg/day ip for 2 days) and a high dose (50 mg/kg/day ip for 1 day) of DEX were
administered to wild-type (WT) and PXR-null mice.
Total RNA Isolation. Total RNA was isolated using RNA-Bee reagent (Tel-Test
Inc., Friendswood, TX) according to the manufacturer's protocol. Total RNA
concentrations were quantified spectrophotometrically at 260 nm, and purity was
confirmed by 260/280 and 260/230 nm ratios. Total RNA was diluted to 1µg of total
RNA per microliter with diethyl pyrocarbonate-treated deionized water. Integrity of RNA
samples was determined by formaldehyde-agarose gel electrophoresis with
visualization by ethidium bromide fluorescence under ultraviolet light.
Branched DNA Signal Amplification Assay. Mouse Ces mRNA expression was
quantified using the branched DNA (bDNA) assay (Quantigene bDNA signal
amplification kit; Panomics, Inc., Fremont, CA). The GenBank accession numbers of all
genes, the sequences, and functions of the probe sets for each mouse CES are shown
in Table 1. Reagents required for RNA analysis were supplied in the Quantigene®
bDNA signal amplification kit (Bayer Diagnostics, East Walpole, MA). The mRNAs were
analyzed as previously described (Hartley and Klaassen, 2000). Data are presented as
relative light units (RLUs) per 8 µg of total RNA.
Multiplex Suspension Array. Mouse Ces mRNA expression from the
chemical treatment experiment involving both WT and knockout mice were determined
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by Mutiplex suspension array (Panomics-Affymetrix, Inc., Fremont, CA). Individual
gene accession numbers can be accessed at www.panomics.com (sets #21086).
Samples were analyzed using a Bio-Plex 200 System Array reader with Luminex 100
xMAP technology, and the data were acquired using Bio-Plex Data Manager version 5.0
(Bio-Rad, Hercules, CA). Assays were performed according to each manufactures’
protocol. RNA data were normalized to glyceraldehydes-3-phosphate dehydrogenase
(Gapdh) mRNA and are presented as relative light units (RLUs).
Statistical Analysis. Data were analyzed by one-way ANOVA, followed by
Duncan's post-hoc test. Statistical significance was set at p < 0.05. Bars represent
mean ±SEM (n=5).
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Results
Regulation of mouse Aadac, Ces1c, 1d, 1e, 1f, and 1g. As shown in Fig 1,
Aadac mRNA was highly expressed in mouse livers, and suppressed by all three AhR
ligands: TCDD (67%), PCB (57%), and BNF (33%); two CAR activators: TCPOBOP
(68%) and DAS (50%); two PXR ligands: PCN (50%) and DEX (63%); as well as the
Nrf2 activator BHA (38%). Ces1c was decreased by the PXR ligand DEX (75%).
Ces1d was decreased 75% by the PXR ligand DEX. Ces1e was decreased about 70%
by the PXR ligand DEX and the Nrf2 activator BHA. Ces1f was decreased by the AhR
ligand BNF (40%), the CAR activator DAS (47%), and the PXR ligand DEX (74%).
Ces1g was decreased about 70% by the PXR ligand DEX, but increased by all three
Nrf2 activators BHA (56%), OPZ (132%), and EXQ (38%).
Regulation of mouse Ces2a, 2b, 2c, 2e, 3a, and 5a. As shown in Fig 2, Ces2a
was increased by all three CAR activators: TCPOBOP (90%), PB (49%), and DAS
(57%); two PXR ligands: PCN (222%) and SPR (177%); as well as the Nrf2 activator
ETH (53%). Ces2b was minimally expressed in livers of mice, and was induced by two
Nrf2 activators OPZ (70%) and ETH (100%). Ces2c was increased markedly by two
CAR activators TCPOBOP (85%) and PB (150%), two PXR ligands PCN (70%) and
SPR (90%), as well as two Nrf2 activators OPZ (140%) and ETH (110%). Ces2e was
minimally expressed in livers of mice, and was not altered by any MEI. Ces3a was
decreased by two AhR ligands: TCDD (75%) and BNF (63%), the CAR activator
TCPOBOP (56%), the PXR ligand DEX (74%), as well as the Nrf2 activator BHA (86%).
Ces5a was minimally expressed in mouse livers, and was not altered by any of the
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MEIs. Due to the minimal expression in liver, the regulation of Ces2b, 2e, and 5a was
not further investigated in the present study.
Effect of TCDD on mRNA expression of Aadac and Ces3a in livers of WT and
AhR-null mice. To determine whether the inhibition of Aadac and Ces3a by TCDD was
dependent on AhR, both WT and AhR-null mice were treated with TCDD. As shown in
Fig 3, Aadac was similarly expressed in control WT and AhR-null mice. TCDD
decreased Aadac about 40% in WT mice, but not in AhR-null mice. Ces3a had lower
expression in AhR-null than WT mice. TCDD suppressed Ces3a approximately 57% in
WT mice, but not in AhR-null mice. Thus, TCDD-mediated inhibition of Aadac and
Ces3a is AhR-dependent.
Effect of TCPOBOP on mRNA expression of Aadac, Ces2a, 2c, and 3a in
livers of WT and CAR-null mice. To determine whether TCPOBOP regulation of
Aadac, Ces2a, 2c, and 3a was dependent on CAR, both WT and CAR-null mice were
treated with TCPOBOP. As shown in Fig 4, Aadac, Ces2a, and 2c had similar
expression in WT and CAR-null mice, whereas Ces3a was lower in CAR-null mice.
Aadac and Ces3a were suppressed by TCPOBOP in WT but not CAR-null mice.
TCPOBOP strongly induced Ces2a and 2c in WT but not CAR-null mice. Taken
together, CAR is required for the TCPOBOP-mediated suppression of Aadac and
Ces3a as well as induction of Ces2a and 2c in livers of mice.
Effect of PCN on mRNA expression of Aadac, Ces2a, and 2c in livers of WT
and PXR-null mice. To determine the role of PXR in PCN-mediated regulation of
Aadac, Ces2a, and 2c, both WT and PXR-null mice were treated with PCN. As shown
in Fig 5, Aadac and Ces2a were similarly expressed in livers of WT and PXR-null mice,
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whereas Ces2c was higher in livers of PXR-null than WT mice. Aadac was decreased
by PCN in WT mice, but not in PXR-null mice. PCN increased Ces2a and 2c in WT
mice, but not in PXR-null mice. Thus, PXR is required for the PCN-mediated
suppression of Aadac as well as the induction of Ces2a and 2c in livers of mice.
Effect of OPZ on mRNA expression of Ces1g and 2c in livers of WT and Nrf2-
null mice. To determine whether the induction of Ces1g and 2c by OPZ was Nrf2
dependent, both WT and Nrf2-null mice were treated with OPZ. Ces1g and 2c mRNA
were lower in Nfr2-null than WT mice (Fig 6). OPZ increased Ces1g and 2c in WT but
not Nrf2-null mice. Thus, OPZ-mediated induction of Ces1g and 2c is Nrf2-dependent.
Effect of Low-dose and High-dose DEX on mRNA expression of Aadac,
Ces1c, 1d, 1e, 1f, 1g, and 3a in livers of WT and PXR-null mice. Both PXR and GR
are shown to be activated by DEX (Shi et al., 2008). Activation of PXR requires
micromolar, whereas activation of GR requires nanomolar concentrations of DEX. In
the present study, WT and PXR-null mice were treated with DEX at both low and high
doses. Aadac, Ces1d, 1e, and 3a were similarly expressed in control WT and control
PXR-null mice (Fig 7). In contrast, PXR-null mice had lower Ces1g but higher Ces1c
and 1f than WT mice. Aadac was suppressed by high-dose, but not low-dose DEX in
WT mice. In contrast, Ces1c, 1d, 1e, 1f, 1g, and 3a were suppressed by both low- and
high-dose DEX in WT mice. Aadac, Ces1g, and 3c were suppressed by high-dose but
not low-dose DEX in PXR-null mice. In contrast, Ces1c, 1d, 1e, and 1f were
suppressed by both low- and high-dose DEX in PXR-null mice. Interestingly, Ces1c, 1d,
1e, and 1f were suppressed more by high-dose than low-dose DEX in PXR-null mice.
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Taken together, PXR does not appear to be involved in the DEX-mediated regulation of
Cess in livers of mice.
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Discussion
The majority of identified CES/Ces enzymes belong to either the CES1 or CES2
subfamily (Satoh and Hosokawa, 2006). CES1 subfamily mainly hydrolyzes substrates
with small alcohol and large acyl groups, whereas CES2 subfamily prefers to hydrolyze
substrates with large alcohol or small acyl groups. The present study demonstrates that
the mRNA expression of the Ces1 subfamily is generally not altered by most MEIs,
whereas the mRNA expression of the Ces2 subfamily is readily induced by ligands of
CAR, PXR, and Nrf2 in livers of mice. This suggests that MEIs increase the liver
hydrolysis of ester-containing drugs, in particular those with large alcohol and small acyl
groups, such as irrinotecan, a carbamate prodrug used in the treatment of colorectal
cancers (Humerickhouse et al., 2000). Of the 11 Cess investigated, Ces2b, 2e, and 5a
are minimally expressed in livers of mice, and were not altered by most of the MEIs,
except that Ces2b is induced by two Nrf2 ligands, OPZ and ETH. Due to the minimal
expression of these Cess in liver, we did not further investigate their regulatory
mechanisms.
AADAC was purified from human liver when studying the metabolism of
carcinogens, and has been reported to be involved in the hydrolysis of various clinical
therapeutic drugs, such as flutamide, phenacetin, and rifamycins (Probst et al., 1994;
Watanabe et al., 2009; Watanabe et al., 2010; Nakajima et al., 2011). In the present
study, mouse liver Aadac mRNA is suppressed by three AhR ligands, two CAR ligands,
two PXR ligands, and one Nrf2 activator. The suppression of Aadac by TCDD,
TCPOBOP, and PCN is dependent on AhR, CAR, and PXR, respectively. Trickett et al.
(Trickett et al., 2001) reported that two PPARα ligands fibrate and Wy-14,643 increase
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Aadac mRNA in the intestine, but not in livers of mice. Consistently, the present finding
demonstrates that Aadac mRNA in livers of mice is not altered by any of three PPARα
ligands.
In general, AhR ligands have little effect on most Cess in livers of mice. It is
reported that the AhR ligand, PCB (Aroclor 1254), tended to decrease rat Ces activities
in both whole liver and liver microsomes (Carr et al., 2002). In contrast, the present
data indicate that PCB has no effect on mouse liver Cess. AhR ligands BNF and TCDD
have been reported to decrease Ces1f mRNA in livers of rats (Morgan et al., 1994;
Yang et al., 2001). In the present study, mouse liver Ces1f mRNA is also suppressed
by BNF, but not altered by TCDD. Interestingly, both BNF and TCDD suppress mouse
liver Ces3a, and the TCDD-mediated suppression of Ces3a is AhR dependent. Ces3a
(or Es31) is expressed predominantly in male mouse livers (Aida et al., 1993). There is
little information on the substrate specificity of CES3/Ces3a. However, the pig homolog
of Ces3a was shown to be involved in the hydrolysis of indomethacin, a drug commonly
used to reduce fever, pain, and swelling (Terashima et al., 1996). Therefore, AhR
ligands in livers of mice may decrease the hydrolytic activity toward substrates such as
indomethacin.
CAR and PXR are two closely related xenobiotic-sensing nuclear receptors,
which are mainly expressed in hepatic tissue. The CAR activator PB has been shown to
induce hepatic microsomal CES activity in mice as well as two Ces isozymes in rat liver
microsomes (Ketterman et al., 1987; Hosokawa et al., 1988). Compared to CAR, a
critical role of PXR in the regulation of CES gene expression in both humans and
rodents has been suggested in many studies. PXR is implicated in the induction of
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human CES2 by 8-methoxypsoralen (Yang and Yan, 2007). PCN markedly induces rat
Ces1e (also known as ES-3, Egasin or Ces RL2) (Hosokawa et al., 1993a). Over-
expression of constitutively active human PXR has a positive effect on Ces gene
expression in livers of mice (Rosenfeld et al., 2003). The present study demonstrates
that both Ces2a and 2c in livers of mice are induced by CAR and PXR ligands, and the
induction by TCPOBOP and PCN is dependent on CAR and PXR, respectively. Xu et al.
also reported that Ces2a is a target gene of both CAR and PXR in livers of mice (Xu et
al., 2009). Therefore, together with numerous other drug-metabolizing enzymes and
drug transporters, CAR and PXR also regulate the expression of key CES enzymes that
coordinately determine the pharmacokinetic and pharmacodynamic properties of drugs
and other xenobiotics.
PPARα is one of three subtypes of PPARs, and is predominantly expressed in
tissues with a high oxidative capacity, such as liver and heart. Feeding clofibrate, a
PPARα ligand, has little effect on Ces1d (also known as TGH) in livers of wild-type or
PPARα-null mice (Dolinsky et al., 2003). This is consistent with the present study.
Poole et al. showed that Ces1f (also known as Es-4) was down-regulated by the
peroxisome proliferator Wy-14,643 (Poole et al., 2001). In the present study, Ces1f
mRNA in livers of male mice tends to be, but not significantly decreased by two PPARα
ligands CLOF and DEHP. Ces1f has higher expression in livers of female than male
mice (Supplemental Fig 2a). Interestingly, the PPARα ligand CPFB decreased Ces1f
mRNA in a PPARα-dependent manner in livers of female, but not male mice
(Supplemental Fig 2b). Feeding 2% DEHP in the diet for 7 days significantly increased
the Ces protein and hydrolytic activity in mouse liver microsomes (Hosokawa et al.,
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1994). Furihata et al. identified that Ces2c (also known as mCES2) is induced by
DEHP (Furihata et al., 2004). In contrast, in the present study, DEHP has little effect on
Cess in livers of mice. This may be due to the shorter-term (4 days) treatment in the
present study. Taken together, PPARα ligands have little effect on most Cess, which is
confirmed by the inducer treatment in PPARα-null mice (Supplemental Fig 3).
Nrf2 is the most important regulator of antioxidant proteins and phase II-enzymes
that protect against oxidative stress and electrophiles in cells. The Nrf2 ligand BHA
induces CES1 in human hepatocytes (Takakusa et al., 2008). Maruichi et al. also
reported that human CES1 (also known as CES1A1) is induced by tert-
butylhydroquinone and sulforaphane in a Nrf2-dependent manner (Maruichi et al., 2009).
Sulforaphane has been shown to moderately induce CES activity in livers of WT, but not
in Nrf2-null mice (Thimmulappa et al., 2002). The Nrf2 activator BHA increases the
microsomal Ces activity in mice ((Ketterman et al., 1987). The present study
demonstrates that the Nrf2 ligand BHA increases Ces1g, but decreases Ces1e and 3a
in mouse livers. Our previous study demonstrated that Ces1g and 2c are lower in Nrf2-
null mice and higher in Keap1-knockdown (Nrf2 activated) than in wilde-type mice
(Reisman et al., 2009). Consistently, the Nrf2 ligand OPZ induces Ces1g and 2c in
mouse livers in a Nrf2-dependent manner. Interestingly, whereas OPZ has no effect on
Ces2a or 3a in livers of male mice, OPZ increases Ces2a and decreases Ces3a in
livers of female WT, but not in female Nrf2-null mice (Supplemental Fig 4).
Species differences have been observed in DEX regulation of CESs. DEX
treatment decreased Ces1d (also known as Es-10) and 1f (also known as Es-4 and
hydrolase B) in rat liver, whereas it slightly increased human CES1 and 2 in cultured
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human hepatocytes (Zhu et al., 2000; Furihata et al., 2005). In the present study, DEX
suppresses almost all the Ces1 isozymes, as well as Ces3a in livers of mice. Both PXR
and GR are known to mediate DEX regulation of rat Cess, and the concentration of
DEX determines the involvement of PXR and/or GR in the suppression of rat Ces1d and
1f (Shi et al., 2008). To determine the role of PXR in DEX suppression of mouse Cess,
both a low-dose (2mg/kg) and a high-dose (50mg/kg) of DEX were treated to WT and
PXR-null mice. Low-dose DEX is proposed to activate the GR, whereas high-dose DEX
is able to activate both GR and PXR. Low-dose DEX tended to, but not significantly,
increase Cyp3a11, whereas high-dose DEX significantly increased Cyp3a11 in livers of
WT mice (Supplemental Fig 5). However, neither low-dose nor high-dose DEX had
effect on Cyp3a11 in livers of PXR-null mice (Supplemental Fig 5). This suggests that
high-dose DEX is able to induce Cy3a11 in a PXR-dependent manner. High-dose DEX
suppressed Aadac, Ces1c, 1d, 1e, 1f, 1g, and 3a in both WT and PXR-null mice,
suggesting that PXR may not be required for the DEX-mediated suppression of Cess in
livers of mice.
Collectively, the current study provides important insights into the chemical
regulation of mouse liver Cess. Cess in livers of mice are shown to be regulated by
AhR, CAR, PXR, Nrf2, and PPARα. Future studies should be performed by using
primary human hepatocytes to determine whether these five signaling pathways are
conserved in humans. To conclude, the present study on the inductive abilities and
regulatory mechanism of MEIs on CESs/Cess will ultimately aid in rational drug design
and the development of novel prodrugs, and will be clinically beneficial to predict drug-
drug interactions and clarify the cause of individual responses to various drugs.
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ACKNOWLEDGMENTS.
The authors would like to thank the members in Dr. Klaassen’s lab for their assistance
in tissue collection and manuscript review.
Authorship Contribution.
Participated in research design: Zhang, Cheng, and Klaassen.
Conducted experiments: Zhang, Cheng, and Aleksunes.
Contributed new reagents or analytic tools: Cheng and Aleksunes.
Performed data analysis: Zhang, Cheng, and Aleksunes.
Wrote or contributed to the writing of the manuscript: Zhang, Cheng, and Klaassen.
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Footnotes: This work was supported by the National Institute of Health [grant
ES009649, ES019487, and ED081461].
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Figure Legends
Figure 1. mRNA expression of Aadac, Ces1c, 1d, 1e, 1f, and 1g in mouse livers
after treatment with prototypical drug-metabolizing enzyme inducers. Total RNA
from livers of treated male C57BL/6 mice (n = 5/treatment) was analyzed by the bDNA
assay. All data are expressed as mean ± S.E. for five mice in each group, except for
control groups, which were combined from the four individual control groups after it was
determined that they were not statistically different. Asterisks (*) indicate statistically
significant differences between control and treated mice (p < 0.05).
Figure 2. mRNA expression of Ces2a, 2b, 2c, 2e, 3a, and 5a in mouse livers after
treatment with prototypical drug-metabolizing enzyme inducers. Total RNA from
livers of treated male C57BL/6 mice (n = 5/treatment) was analyzed by the bDNA assay.
All data are expressed as mean ± S.E. for five mice in each group, except for control
groups, which were combined from the four individual control groups after it was
determined that they were not statistically different. Asterisks (*) indicate statistically
significant differences between control and treated mice (p < 0.05).
Figure 3. Effect of TCDD on mRNA expression of Aadac and Ces3a in livers of
WT and AhR-null mice. Total RNA from livers of control and TCDD-treated male
C57BL/6 and AhR-null mice (n = 5/group) was analyzed by the Quantigene Plex assay.
The data are reported as ratio of carboxylesterase mRNA to GAPDH mRNA expression
per 500ng of total RNA. All data were expressed as mean ± S.E. Asterisks (*) indicate
statistically significant differences between control and treated mice of the same
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genotype (p<0.05). Dagges (#) indicate statistically significant differences between
control WT and control null mice (p<0.05).
Figure 4. Effect of TCPOBOP on mRNA expression of Aadac, Ces2a, 2c, and 3a in
livers of WT and CAR-null mice. Total RNA from livers of control and TCPOBOP-
treated male C57BL/6 and CAR-null mice (n = 5/group) was analyzed by the
Quantigene Plex assay. The data are reported as ratio of carboxylesterase mRNA to
GAPDH mRNA expression per 500ng of total RNA. All data were expressed as mean ±
S.E. Asterisks (*) indicate statistically significant differences between control and
treated mice of the same genotype (p<0.05). Dagges (#) indicate statistically significant
differences between control WT and control null mice (p<0.05).
Figure 5. Effect of PCN on mRNA expression of Aadac, Ces2a, and 2c in livers of
WT and PXR-null mice. Total RNA from livers of control and PCN-treated male
C57BL/6 and PXR-null mice (n = 5/group) was analyzed by the Quantigene Plex assay.
The data are reported as ratio of carboxylesterase mRNA to GAPDH mRNA expression
per 500ng of total RNA. All data were expressed as mean ± S.E. Asterisks (*) indicate
statistically significant differences between control and treated mice of the same
genotype (p<0.05). Dagges (#) indicate statistically significant differences between
control WT and control null mice (p<0.05).
Figure 6. Effect of OPZ on mRNA expression of Ces1g and 2c in livers of WT and
Nrf2-null mice. Total RNA from livers of control and OPZ-treated male C57BL/6 and
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Nrf2-null mice (n = 5/group) was analyzed by the Quantigene Plex assay. The data are
reported as ratio of carboxylesterase mRNA to GAPDH mRNA expression per 500ng of
total RNA. All data were expressed as mean ± S.E. Asterisks (*) indicate statistically
significant differences between control and treated mice (p< 0.05). Dagges (#) indicate
statistically significant differences between control WT and control null mice (p<0.05).
Figure 7. Effect of Low-dose (2 mg/kg) and High-dose (50 mg/kg) DEX on mRNA
expression of Aadac, Ces1c, 1d, 1e, 1f, 1g, and 3a in livers of WT and PXR-null
mice. Wild-type and PXR-null mice were administered with both a low dose (2
mg/kg/day ip for 2 days) and a high dose (50 mg/kg/day ip for 1 day) of DEX. Total
RNA from liver of control and DEX-treated male C57BL/6 and male PXR-null mice (n =
5/group) was analyzed by the bDNA assay. The data are reported as the mRNA fold
change to the carboxylesterase mRNA of control WT mice. All data were expressed as
mean ± S.E. Single asterisks (*) indicate statistically significant differences between
treated and control mice of the same genotype (p<0.05). Double asterisks (**) indicate
statistically significant differences between low-dose and high-dose DEX-treated mice
(p<0.05). Dagges (#) indicate statistically significant differences between control WT
and control null mice (p<0.05).
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Table 1. Oligonucleotide probes generated for analysis of mouse Ces mRNA
expression by Quantigene branched DNA signal amplification assay
Functiona Probe Sequence Function Probe Sequence
Ces1c (NM_007954) Ces1e (Cont’d) CE ccagttggtccaggtgagccTTTTTctcttggaaagaaagt LE caatcacagatggtacaccgaataTTTTTaggcataggacccgtgtct
CE cccctgatgactctccaaagatTTTTTctcttggaaagaaagt BL Aaaagattcaattttagtgtggtcc
CE agaggtccttgcccagaggaTTTTTctcttggaaagaaagt BL Tccatcaagcacagtgggaac
CE cagcctgtatattcttcttgcccTTTTTctcttggaaagaaagt BL Ggcatctttggcagcaacac
LE ggttactaccaccacgttctcatgTTTTTaggcataggacccgtgtct BL Tgtagggcactgtgttgaagttc
LE tcacctgtgctaaataatccccTTTTTaggcataggacccgtgtct BL Cgtcatctggtccaattttacatca
LE cagtttcctgggctgtgttcaTTTTTaggcataggacccgtgtct BL Ctcagggaggttaagaagaaaaga
LE ggtcacggaatccgggttcTTTTTaggcataggacccgtgtct BL Gtgtaatctttatctcttaaatacttctcaa
LE gataagacaaggacagagacactgatacTTTTTaggcataggacccgtgtct BL Agaagttggtctttatttctgcct
LE acgtttgtatttatgaccacaccaTTTTTaggcataggacccgtgtct Ces1f (NM_144930)
LE gcagctgatgaggtgtcattacactTTTTTaggcataggacccgtgtct CE tctttggggtgatgagaagcttTTTTTctcttggaaagaaagt
LE gcgcaagcactgaaccatgTTTTTaggcataggacccgtgtct CE attgatctcctcttcggaggcTTTTTctcttggaaagaaagt
BL Aaatacccaggcggtattgaat CE tcttggcttctctggtataagccTTTTTctcttggaaagaaagt
BL Tggacccagcgtagtgcag LE gggtagtattgatactcatacatgtaggtTTTTTaggcataggacccgtgtct
BL Cctccaaagtttgcaatgttatct LE ttgccattagggttcccattTTTTTaggcataggacccgtgtct
BL Ctctcagaaatggctctgtgga LE aagatatccttctttctgatcatactttTTTTTaggcataggacccgtgtct
BL Gggaaagagtagctattatttcattca LE tgggtggtgccaccaatatgTTTTTaggcataggacccgtgtct
Ces1d (NM_053200) b LE tggcagagctttgcattcacTTTTTaggcataggacccgtgtct
CEc tctgggctgcctgagttgagTTTTTctcttggaaagaaagt LE tcccttccctgggctctgTTTTTaggcataggacccgtgtct
CE cctctgggctgactccttggTTTTTctcttggaaagaaagt BL Atggtctcctactacattcttggg
CE gaaggagataaatatttcctttttaatgTTTTTctcttggaaagaaagt BL Cgaagacagagtagacatcatctgc
LE cttctggtcatattctggccagtTTTTTaggcataggacccgtgtct BL Accctctcttaaaattggagcac
LE gcaccaatcttcagatacccttcTTTTTaggcataggacccgtgtct BL Gaatttcatcaccatcttgctgag
LE actcacttctttgtccttcagccTTTTTaggcataggacccgtgtct BL Ccgagcaaagttggccca
LE acatgttccctgtgggatggTTTTTaggcataggacccgtgtct BL Ccttcagtctctgggcttgc
LE tgaccctcctgatcagagctcaTTTTTaggcataggacccgtgtct BL Gtgtccagaaagtcacttcctctt
LE gactccaggttcttaagcacagcTTTTTaggcataggacccgtgtct BL Ggttgtttcttggcaagggact
BL ccctgagctcagcccaaaa BL Agctcattgtggtatggctgg
BL aaatcttctgtggaataatactccttt BL Acatacactgttagtggaagaaccata
BL cttcaaagataagtgttatttttctacaa BL Cctccaagatccatcttggattt
BL catttgtatgaaataccatataatgttatag BL Cctatatctatgacaaagttcttcaggat
BL tcaaaacattattttattttttacaagtt BL Ggccaatgaggcagccct
Ces1e (NM_133660) Ces1g (BC026897)
CE aaagggatggctctgtctggaTTTTTctcttggaaagaaagt CE cccaaatttcattttcagtgagaTTTTTctcttggaaagaaagt
CE ttctcagccaggatctcctcaTTTTTctcttggaaagaaagt CE ggaggaagggatagctctctctgTTTTTctcttggaaagaaagt
CE ggcagaatccagccaaactcTTTTTctcttggaaagaaagt CE cagcttttcagagatgttaagaattgTTTTTctcttggaaagaaagt
LE gtctccatgcaaatccagcttaTTTTTaggcataggacccgtgtct LE gcaacactccatcaatcacagtagTTTTTaggcataggacccgtgtct
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LE ttgcttgttgattcccacgaTTTTTaggcataggacccgtgtct LE gccaaactcatgcttgttgatgTTTTTaggcataggacccgtgtct
LE gagggtgggtagttcatcatagttTTTTTaggcataggacccgtgtct LE gatgcagctgtcttctggtccTTTTTaggcataggacccgtgtct
LE ggacttcttcaagagagacatggcTTTTTaggcataggacccgtgtct LE ggtaggcctgccacaggatgTTTTTaggcataggacccgtgtct
LE tggccactgcaattgcatcTTTTTaggcataggacccgtgtct LE gcagggtcttctgtccctcctaTTTTTaggcataggacccgtgtct
LE ccacatccccaatcaattccTTTTTaggcataggacccgtgtct LE ccaggaacaggtctgtcattgtgTTTTTaggcataggacccgtgtct
Ces1g (Cont’d) Ces2b (Cont’d)
LE ccgaacataatgtctccaatcaagtTTTTTaggcataggacccgtgtct BL accagggcctctgagtccat
BL Cctccaagagctcatcttcagtct BL ttaatagccagaatctctgcttcac
BL Gggtctccaagaaaatcaacagt BL gggatcatctggacaagcttg
BL Ttgaaactcttctcagccagaatc BL ttgggatgcctggggaag
BL Cccaccatgtaggggacagtg BL atgctggggacagggtgaa
BL Ccaaaaacattggaatgatcca BL aggttctctctggttatttcctttat
BL Agttttctttcagagagtgggaagt Ces2c (NM_145603)
BL Aatacttttcaatagctgctggaat CE gcaggttagagccctcatggTTTTTctcttggaaagaaagt
BL Tcttctggtgcctttggca CE tctggcatgctggtctccagTTTTTctcttggaaagaaagt
Ces2a (NM_133960) CE cgtagggcagctgcttggtTTTTTctcttggaaagaaagt
CE agctcctcctcctcagtgaggTTTTTctcttggaaagaaagt LE tgttgagatacaggcagtcctcaTTTTTaggcataggacccgtgtct
CE catttcttgcgaaattggccTTTTTctcttggaaagaaagt LE ccatggatccacaccatcacagTTTTTaggcataggacccgtgtct
CE gaactgcagccttctggccTTTTTctcttggaaagaaagt LE ccaccaagtcctcattgactgtcTTTTTaggcataggacccgtgtct
CE ctctgcatgcttgtcctgagaaTTTTTctcttggaaagaaagt LE cccagacgatactggatagtaacaaTTTTTaggcataggacccgtgtct
LE tccaaattcatgtcccacaaataTTTTTaggcataggacccgtgtct LE ccaggtatccccagttgccTTTTTaggcataggacccgtgtct
LE caggtactgctcatcatggtccTTTTTaggcataggacccgtgtct LE cgatgttctgctggacccagTTTTTaggcataggacccgtgtct
LE caggctgggtgtccagctgTTTTTaggcataggacccgtgtct BL Ccattcctataaccagtgcacct
LE ttcagggctcgacccacagTTTTTaggcataggacccgtgtct BL Aatagagatccatcaaacatggaag
LE ccctgacacaggacgctacagTTTTTaggcataggacccgtgtct BL Tgctgaaaaagcccaggaca
LE atctgcttttaatctcacacaccttTTTTTaggcataggacccgtgtct Ces2e (NM_172759) LE ggactttgtaacctcagaattacaccTTTTTaggcataggacccgtgtct CE tgattatttctgttattgagttacaggtcTTTTTctcttggaaagaaagt
BL Cagtacttcatcatcatcctctttagc CE ggaaaggatgcgatttcctctTTTTTctcttggaaagaaagt
BL Ccctcactgttggggttcc CE attcttggccttgcttgaggTTTTTctcttggaaagaaagt
BL Aacacaggccaggagggtaga LE ttgctatgttgtccatggtggtTTTTTaggcataggacccgtgtct
BL Ggggcagagtcttggtcca LE gtttgcaagttaccccagctagTTTTTaggcataggacccgtgtct
BL Cccttgagctcctggatcttct LE ccacagaactttacatacatttctgttTTTTTaggcataggacccgtgtct
Ces2b (BC015286) LE cattttctcaccctctatttctcataTTTTTaggcataggacccgtgtct
CE aatgttctgctggatccagcTTTTTctcttggaaagaaagt LE gacatcagaggtctagtagctgagcTTTTTaggcataggacccgtgtct
CE cactctccatgatggctccatTTTTTctcttggaaagaaagt LE atagggacaatggacacagtttttTTTTTaggcataggacccgtgtct
CE tgtttgagcagagcccatgaTTTTTctcttggaaagaaagt LE gcctgctggtcaacccccTTTTTaggcataggacccgtgtct
LE gtagggcagccacttgatccTTTTTaggcataggacccgtgtct BL Cctgaaagactgtatggctcaagtt
LE tgtgccacctgcagactcgTTTTTaggcataggacccgtgtct BL Agtagctggaatgttcagtggct
LE ggcctcacatccagagagcttTTTTTaggcataggacccgtgtct BL Ttcacaaaagtagataatggcaattt
LE ttttgcctctcagacagcgcTTTTTaggcataggacccgtgtct BL Tgtctataaacataccaagtcaagctt
LE aactctccatccaccacagcaTTTTTaggcataggacccgtgtct Ces3a (BC061004)
LE aatcctcagatgccaacaactctTTTTTaggcataggacccgtgtct CE cccaggctggcttgaacttTTTTTctcttggaaagaaagt
LE actcatcgttgttgacaccaatgTTTTTaggcataggacccgtgtct CE tctgggaaggccaggaggTTTTTctcttggaaagaaagt
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LE ccacaggaatggtccaaccaaTTTTTaggcataggacccgtgtct CE gcaaactggctccattgggTTTTTctcttggaaagaaagt
LE ttgtattcttcaaaacagcctgcTTTTTaggcataggacccgtgtct LE cagaggaatggtcagccttcaTTTTTaggcataggacccgtgtct
BL gtttgcctccaaagtgggc LE cctccaaaaacaaaggcattctTTTTTaggcataggacccgtgtct
BL ccaaaaatagtgacccggtcag LE ccatcatggtcaggctcagcTTTTTaggcataggacccgtgtct
BL ggacacaacatgggaagacacact LE cattgggatttcctgtgcgtTTTTTaggcataggacccgtgtct
BL ggaagagtcctttggacatggg LE aggaggcagccccttgcTTTTTaggcataggacccgtgtct
BL aggaagcagggccaccc LE tctagaccaatctccaagtattgttctTTTTTaggcataggacccgtgtct
BL atctcagaggtgtcagtgataaggta LE cttcaccccagtccgtggtTTTTTaggcataggacccgtgtct
BL ggccaccgtagtggagacc LE aactgtagccgacccttctttagTTTTTaggcataggacccgtgtct
Ces3a (Cont’d) Ces5a (Cont’d)
BL Tgcaaaagaactgggtgtatgc BL Agagcagtagactcattggagcc
BL gaactctcatcagtgaggaaagga BL Tgcagcaaagtatgtatcaaggtg
BL Tgcttctcttcctctgtggcc BL Catggaagtactctttagtcacaatgt
BL Aactggtttaattggggcca BL Aagtccagtaaagtgtctcggatg
Ces5a (AB186393) Aadac (NM_023383) CE aagaagacttttccctcaggcaTTTTTctcttggaaagaaagt CE cgcctgagagccattgactttTTTTTctcttggaaagaaagt
CE tcgagtgaatgattttgctttctTTTTTctcttggaaagaaagt CE tgggctgtccatctcgatagagTTTTTctcttggaaagaaagt
CE tcctggttattgactccaatgatagTTTTTctcttggaaagaaagt CE ccatcgtaaactacgatacacatcttcTTTTTctcttggaaagaaagt
CE tcggtcggagaatgcttgcTTTTTctcttggaaagaaagt LE cttttgggtatgtatatgcggacTTTTTaggcataggacccgtgtct
LE gcaaccacctgcaaatcatgTTTTTaggcataggacccgtgtct LE caccatgaatataaaacaagcccTTTTTaggcataggacccgtgtct
LE tgatacattgcaatcacaaacatttTTTTTaggcataggacccgtgtct LE ctccccaaacaccagccacTTTTTaggcataggacccgtgtct
LE ggctaaggctcatcaactccaTTTTTaggcataggacccgtgtct LE tgacacaacaacagcatcaagtttaTTTTTaggcataggacccgtgtct
LE aaggtactattttcaatgtcttttgagTTTTTaggcataggacccgtgtct LE gactctcctaggatctacaccatatttTTTTTaggcataggacccgtgtct
LE acgggcaatatgtagccacacTTTTTaggcataggacccgtgtct LE cagcactgtctccagacacaccTTTTTaggcataggacccgtgtct
LE acaaatgctgggtggggataTTTTTaggcataggacccgtgtct LE agctgcggctaagtttccacTTTTTaggcataggacccgtgtct
BL Tttcagatcattatcagagctcttca BL Tgtcataggagaagtgagcagca
BL Cttcagcaaagccttggagtc BL Tggggctaagccatagtcagt
BL Agaagaaagaaccgtcgaccac BL Aaactgtcttggaaaatggtgttt
BL Acaacagttctagtggttcttcgg BL Ttcaaggacgtcctcttgtaagaa
BL Gaggagaatctcaggagtgtctctc
Note:
aFunction refers to the type of bDNA oligonucleotide probe represented by each sequence.
bGenBank accession numbers for each transcript are given in cell under the gene name.
cCE, capture extender; LE, label extender; BL, blocker.
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This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on March 19, 2012 as DOI: 10.1124/dmd.111.043877
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This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on March 19, 2012 as DOI: 10.1124/dmd.111.043877
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This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on March 19, 2012 as DOI: 10.1124/dmd.111.043877
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This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on March 19, 2012 as DOI: 10.1124/dmd.111.043877
at ASPE
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This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on March 19, 2012 as DOI: 10.1124/dmd.111.043877
at ASPE
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This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on March 19, 2012 as DOI: 10.1124/dmd.111.043877
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