Functional characterization of human and cynomolgus monkey cytochrome P450 2E1 enzymes

Article abstract of DOI:10.1016/j.lfs.2007.09.002

Cytochrome P450 2E1 (CYP2E1) is an enzyme of major toxicological interest because it metabolizes various drugs, precarcinogens and solvents to reactive metabolites. In this study, human and cynomolgus monkey CYP2E1 cDNAs (humCYP2E1 and monCYP2E1, respectively) were cloned, and the corresponding proteins were heterologously expressed in yeast cells to identify the functions of primate CYP2E1s. The enzymatic properties of CYP2E1 proteins were characterized by kinetic analysis of chlorzoxazone 6-hydroxylation and 4-nitrophenol 2-hydroxylation. humCYP2E1 and monCYP2E1 enzymes showed 94.3% identity in their amino acid sequences. The functional CYP content in yeast cell microsomes expressing humCYP2E1 was 38.4 pmol/mg protein. The level of monCYP2E1 was 42.7% of that of humCYP2E1, although no significant differences were statistically observed. The K_m values of microsomes from human livers and yeast cells expressing humCYP2E1 for CYP2E1-dependent oxidation were 822 and 627 μM for chlorzoxazone 6-hydroxylation, and 422 and 514 μM for 4-nitrophenol 2-hydroxylation, respectively. The K_m values of microsomes from cynomolgus monkey livers and yeast cells expressing monCYP2E1 were not significantly different from those of humans in any enzyme source. V_max and V_max / K_m values of human liver microsomes for CYP2E1-dependent oxidation were 909 pmol/min/mg protein and 1250 nl/min/mg protein for chlorzoxazone 6-hydroxylation, and 1250 pmol/min/mg protein and 2990 nl/min/mg protein for 4-nitrophenol 2-hydroxylation, respectively. The kinetic parameter values of cynomolgus monkey livers were comparable to or lower than those of human liver microsomes (49.5-102%). In yeast cell microsomes expressing humCYP2E1, V_max and V_max / K_m values for CYP2E1-dependent oxidation on the basis of CYP holoprotein level were 170 pmol/min/pmol CYP and 272 nl/min/pmol CYP for chlorzoxazone 6-hydroxylation, and 139 pmol/min/pmol CYP and 277 nl/min/pmol CYP for 4-nitrophenol 2-hydroxylation, respectively, and the kinetic parameters of monCYP2E1 exhibited similar values. These findings suggest that human and cynomolgus monkey CYP2E1 enzymes have high homology in their amino acid sequences, and that their enzymatic properties are considerably similar. The information gained in this study should help with in vivo extrapolation and to assess the toxicity of xenobiotics.

Full text of DOI:10.1016/j.lfs.2007.09.002

Available online at www.sciencedirect.com
Life Sciences 81 (2007) 1436–1445
www.elsevier.com/locate/lifescie
Functional characterization of human and cynomolgus monkey cytochrome
P450 2E1 enzymes
a,1
a,1
a
b
Nobumitsu Hanioka , Maki Yamamoto , Hiroyuki Iwabu , Hideto Jinno ,
b
c
d
a
Toshiko Tanaka-Kagawa , Shinsaku Naito , Takefumi Shimizu , Kazufumi Masuda ,
a
a,
⁎
Takashi Katsu , Shizuo Narimatsu
a
Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 1-1-1 Tsushima-naka, Okayama 700-8530, Japan
b
Division of Environmental Chemistry, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan
c
Division of Pharmacology, Drug Safety and Metabolism, Otsuka Pharmaceutical Factory Inc., 115 Tateiwa Muya-cho, Naruto 772-8601, Japan
d
Department of Metabolism and Analytical Research, Ina Research Inc., 2148-188 Nishiminowa, Ina 399-4501, Japan
Received 16 June 2007; accepted 5 September 2007
Abstract
Cytochrome P450 2E1 (CYP2E1) is an enzyme of major toxicological interest because it metabolizes various drugs, precarcinogens and
solvents to reactive metabolites. In this study, human and cynomolgus monkey CYP2E1 cDNAs (humCYP2E1 and monCYP2E1, respectively)
were cloned, and the corresponding proteins were heterologously expressed in yeast cells to identify the functions of primate CYP2E1s. The
enzymatic properties of CYP2E1 proteins were characterized by kinetic analysis of chlorzoxazone 6-hydroxylation and 4-nitrophenol 2-
hydroxylation. humCYP2E1 and monCYP2E1 enzymes showed 94.3% identity in their amino acid sequences. The functional CYP content in
yeast cell microsomes expressing humCYP2E1 was 38.4 pmol/mg protein. The level of monCYP2E1 was 42.7% of that of humCYP2E1,
although no significant differences were statistically observed. The K
humCYP2E1 for CYP2E1-dependent oxidation were 822 and 627 μM for chlorzoxazone 6-hydroxylation, and 422 and 514 μM for 4-nitrophenol
-hydroxylation, respectively. The Km values of microsomes from cynomolgus monkey livers and yeast cells expressing monCYP2E1 were not
significantly different from those of humans in any enzyme source. Vmax and Vmax /K values of human liver microsomes for CYP2E1-dependent
oxidation were 909 pmol/min/mg protein and 1250 nl/min/mg protein for chlorzoxazone 6-hydroxylation, and 1250 pmol/min/mg protein and
990 nl/min/mg protein for 4-nitrophenol 2-hydroxylation, respectively. The kinetic parameter values of cynomolgus monkey livers were
comparable to or lower than those of human liver microsomes (49.5–102%). In yeast cell microsomes expressing humCYP2E1, Vmax and Vmax
values for CYP2E1-dependent oxidation on the basis of CYP holoprotein level were 170 pmol/min/pmol CYP and 272 nl/min/pmol CYP for
m
values of microsomes from human livers and yeast cells expressing
2
m
2
/
K
m
chlorzoxazone 6-hydroxylation, and 139 pmol/min/pmol CYP and 277 nl/min/pmol CYP for 4-nitrophenol 2-hydroxylation, respectively, and the
kinetic parameters of monCYP2E1 exhibited similar values. These findings suggest that human and cynomolgus monkey CYP2E1 enzymes have
high homology in their amino acid sequences, and that their enzymatic properties are considerably similar. The information gained in this study
should help with in vivo extrapolation and to assess the toxicity of xenobiotics.
©
2007 Elsevier Inc. All rights reserved.
Keywords: Cytochrome P450 (CYP); CYP2E1; Chlorzoxazone 6-hydroxylation; 4-Nitrophenol 2-hydroxylation; Primates; Human; Cynomolgus monkey
Introduction
Cytochrome P450 (CYP) enzymes are hemoproteins that are
responsible for the oxidative metabolism of exogenous
chemicals such as drugs and environmental chemicals, as well
as endogenous substances such as steroids and fatty acids
(Gonzalez, 1990; Rendic and Di Carlo, 1997). They comprise a
⁎
Corresponding author. Tel./fax: +81 86 251 7943.
E-mail addresses: hanioka@pharm.okayama-u.ac.jp (N. Hanioka),
shizuo@pharm.okayama-u.ac.jp (S. Narimatsu).
1
These authors contributed equally to this work.
0024-3205/$ - see front matter © 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.lfs.2007.09.002

N. Hanioka et al. / Life Sciences 81 (2007) 1436–1445
1437
Table 1
superfamily of related enzymes that are grouped into families
and subfamilies based on similarities in amino acid or
nucleotide sequences (Nelson et al., 1996). Most of the
exogenous chemicals have been shown to be oxidized mainly
by CYP1–3 families, and individual CYP isoforms have
considerable, but overlapping, substrate specificities (Gonzalez,
Primers used for construction of OR, humCYP2E1 and monCYP2E1 expression
plasmids
Primer name
Sequence
OR-F
OR-R
5′-CCCAAGCTTGGGAAAAAAATGGGAGACTCC-3′
5′-GGGGTACCCCCTAGCTCCACACGTCC-3′
5′-AAGCTTAAAAAAATGTCTGCCCTCGGAGTG-3′
5′-AAGCTTTCATGAGCGGGGAATGACACA-3′
5′-TTCCTTTCCACAGGATTGTCCTCCCGG-3′
5′-GATTCTGGGGGTCCTCCTCACACTCTA-3′
5′-AAGCTTAAAAAAATGTCTGCCCTCGGAGTC-3′
5′-AAGCTTTCATGAACGGGGAATGACACAG-3′
humCYP2E1-F
humCYP2E1-R
monCYP2E1-F1
monCYP2E1-R1
monCYP2E1-F2
monCYP2E1-R2
1
990; Nelson et al., 1996; Rendic and Di Carlo, 1997).
The human CYP2E1 gene is located in the 10q24.3-qter
region of chromosome 10, and spans 11,413 base pairs with
nine exons, eight introns and typical TATA box (Umeno et al.,
1
988; Nelson et al., 1996). CYP2E1 enzyme consists of 493
amino acid residues and is expressed mainly in the liver
Ingelman-Sundberg et al., 1993; Nelson et al., 1996). CYP2E1
Underlined letters indicate restriction enzyme sites. Italic letters indicate the
yeast consensus sequence.
(
as well as other isoforms of CYP1–3 families has broad
substrate specificity; more than 80 low-molecular hydrophobic
chemicals have been identified as substrates for this CYP
isoform (Guengerich et al., 1991; Rendic and Di Carlo, 1997).
CYP2E1 has also been reported to activate many environmental
chemicals such as benzene, toluene and N-nitrosamines, and
several drugs such as acetaminophen and chlorzoxazone to
highly reactive metabolites (Guengerich et al., 1991; Lieber,
monkey CYP2E1 enzymes is an important aspect of drug
metabolism research.
The purpose of this study was to examine the similarities and
differences in enzymatic properties of CYP2E1 between
humans and cynomolgus monkeys. To achieve this, human
and cynomolgus monkey CYP2E1 cDNAs were cloned, and the
corresponding CYP2E1 proteins were heterologously expressed
in yeast cells. The enzymatic properties of CYP2E1 proteins
were examined by kinetic analysis using chlorzoxazone and 4-
nitrophenol as substrates.
1
997). CYP2E1 activity is mediated by various pathophysio-
logical conditions such as diabetes, obesity and starvation, and
also by a number of environmental factors (Gonzalez, 1990;
Lucas et al., 1998; McCarver et al., 1998). The expression levels
of hepatic CYP2E1 vary by orders of magnitude in humans and
are found to be elevated in alcoholics (Perrot et al., 1989;
Ingelman-Sundberg et al., 1993). Animal studies of CYP2E1
regulation have been found to be very complex, involving
aspects of developmental and hormonal regulation, transcrip-
tional and posttranscriptional control, and protein stabilization
Materials and methods
Materials
Three individual human liver microsomes (one man, 77 years
old; two women, 54 and 56 years old) were purchased from BD
Biosciences (San Jose, CA). Three male cynomolgus monkey
livers (4 years old, 2.7–2.9 kg) were supplied by Ina Research
Inc. (Ina, Japan). The use of human and cynomolgus monkey
livers for this study was approved by the ethics review board of
Okayama University. The RNeasy mini kit, QIAshredder and
MinElute Gel Extraction kit were purchased from Qiagen
(Hilden, Germany); the RNA PCR kit v3.0 and HindIII were
from Takara Bio (Ohtsu, Japan); Taq DNA polymerase and
pGEM-T vector were from Promega (Madison, WI); KOD-plus
DNA polymerase was from Toyobo (Osaka, Japan); BigDye
terminator cycle sequencing reaction kit v3.1 was from Applied
Biosystems (Foster City, CA); pYES3/CT and pYES2/CT yeast
expression vectors, and INVSc1 yeast strain were from
Invitrogen (Carlsbad, CA); yeast nitrogen base was from BD
Diagnostics (Franklin Lakes, NJ); Zymolyase 100T was from
Seikagaku Corporation (Tokyo, Japan); cytochrome c from
horse heart and chlorzoxazone were from Sigma-Aldrich (St.
Louis, MO); 6-hydroxychlorzoxazone was from Daiichi Pure
Chemicals (Tokyo, Japan); 4-nitrophenol, 4-nitrocatechol and
salicylamide were from Nacalai Tesque (Kyoto, Japan);
(
1
Gonzalez, 1990; Yang et al., 1990; Ingelman-Sundberg et al.,
993). Furthermore, it has been suggested that there are large
inter-individual variations in the protein level and catalytic
activity of CYP2E1 in human liver microsomes (Yoo et al.,
1
988; Shimada et al., 1994). Previous in vivo studies of a
Caucasian population using chlorzoxazone as a probe drug
reported a several-fold variation in clearance with a normal
distribution of activities, and suggested that such variation is
caused in part by the genetic polymorphism of CYP2E1 (Kim
and O'Shea, 1995; Lucas et al., 1995).
The CYP2E subfamily has been shown to have only a single
isoform in most mammals, and CYP2E1 cDNAs of humans,
monkeys, rabbits, rats and mice were cloned (Nelson et al.,
1
996). In general, nonhuman primates such as rhesus monkeys
(
Macaca mulatta) and cynomolgus monkeys (Macaca fascicu-
laris) are regarded as experimental animals closer to humans in
studies on safety evaluation and biotransformation for drug
development. Rhesus monkey CYP2E1 cDNA has been cloned
(
GenBank accession number, AY635465); however, the func-
tions of protein encoded by cDNA have not been characterized.
Although cynomolgus monkey CYP2E1 cDNA, which is
presumed to lack N-terminal 45 residues as compared with
the sequence for human CYP2E1, has been isolated from a liver
DNA library (Komori et al., 1992; GenBank accession number,
S55205), the complete cDNA has not been cloned yet.
Furthermore, examination of the functional characterization of
+
NADPH, NADP , glucose 6-phosphate and glucose 6-phos-
phate dehydrogenase were from Oriental Yeast (Tokyo, Japan);
rabbit anti-human CYP2E1 antibody was from Novus Biologi-
cals (Littleton, CO); peroxidase-conjugated goat anti-rabbit
immunoglobulin was from Zymed Laboratories (South San
Francisco, CA); and enhanced chemiluminescence-plus

1438
N. Hanioka et al. / Life Sciences 81 (2007) 1436–1445
reagents were from GE Healthcare Bio-Sciences (Little
Chalfont, UK). All other chemicals and reagents used were of
the highest quality commercially available.
94 °C for 120 s and at 68 °C for 300 s, respectively. The
resulting PCR product was isolated and purified by agarose
electrophoresis. The PCR product was introduced into the
pGEM-T vector after A-tailing and sequenced in both forward
and reverse directions. A DNA fragment corresponding to
humCYP2E1 was cut from the pGEM-T plasmid with HindIII,
and subsequently subcloned into the pYES2/CT expression
vector digested with HindIII (pYES2/humCYP2E1). The
expression plasmids were sequenced to verify the correct
orientation with respect to the promoter for pYES2/CT.
Construction of OR, humCYP2E1 and monCYP2E1 expression
plasmids
Full-length cDNAs encoding human NADPH-cytochrome
P450 reductase (OR), human CYP2E1 (humCYP2E1) and
cynomolgus monkey CYP2E1 (monCYP2E1) were amplified
by polymerase chain reaction (PCR) using the forward and
reverse primers listed in Table 1. The restriction enzyme sites
Total RNA was extracted from cynomolgus monkey liver
using an RNeasy mini kit and QIAshredder according to the
manufacturer's instructions. The total RNA was reverse-
transcribed to cDNA using RNA PCR kit v3.0. The full-length
cDNA encoding monCYP2E1 was amplified by PCR from
single-strand cDNA templates using a monCYP2E1-F1 and
monCYP2E1-R1 primer pair. These primers were designed
based on the nucleotide sequence in the flanking regions of
humCYP2E1 cDNA (GenBank accession number, J02843).
The PCR reaction mix contained 1×PCR buffer, 0.2 mM
dNTPs, each primer at 0.5 μM and 1 U KOD-plus DNA
polymerase in a final volume of 50 μl. PCR consisted of
35 cycles with denaturation at 94 °C for 15 s, annealing at 61 °C
for 30 s and extension at 68 °C for 90 s. Initial denaturation and
final extension were at 94 °C for 120 s and at 68 °C for 300 s,
respectively. The resulting PCR product was isolated and
purified by agarose electrophoresis, and the 5′- and 3′-ends of
the coding region were sequenced in both the forward and
reverse directions. The full-length cDNA thus obtained was
modified by PCR amplification with monCYP2E1-F2 and
monCYP2E1-R2 as primers. The PCR reaction mix contained
1×PCR buffer, 0.2 mM dNTPs, each primer at 0.5 μM and 1 U
KOD-plus DNA polymerase in a final volume of 50 μl. PCR
consisted of 30 cycles with denaturation at 94 °C for 15 s,
annealing at 57 °C for 30 s and extension at 68 °C for 90 s.
Initial denaturation and final extension were at 94 °C for 120 s
and at 68 °C for 300 s, respectively. The resulting PCR product
was isolated and purified by agarose electrophoresis. The PCR
product was introduced into the pGEM-T vector after A-tailing
and sequenced in both forward and reverse directions. A DNA
fragment corresponding to monCYP2E1 was cut from the
pGEM-T plasmid with HindIII, and subsequently subcloned
into pYES2/CT expression vector digested with HindIII
(pYES2/monCYP2E1). The expression plasmids were se-
quenced to verify the correct orientation with respect to the
promoter for pYES2/CT.
(
HindIII for OR-F, humCYP2E1-F, humCYP2E1-R, mon-
CYP2E1-F2 and monCYP2E1-R2, and KpnI for OR-R) were
introduced to the 5′-end of the start codon and the 3′-end of the
stop codon to facilitate subcloning into pYES3/CT or pYES2/
CT expression vector. Yeast consensus sequences (Romanos et
al., 1992) were also introduced upstream of the start codon in
OR-F, humCYP2E1-F and monCYP2E1-F2 to achieve a high
expression of protein in yeast cells.
To achieve the stable expression of OR in yeast cells,
pYES3/CT expression vector was used. The full-length cDNA
encoding OR was amplified by PCR from OR cDNA cloned
into pGEM-3Z vector (Narimatsu et al., 2006) as a template
using OR-F and OR-R primer pair. The PCR reaction mix
contained 1×PCR buffer, 0.2 mM dNTPs, each primer at
0
.5 μM and 1 U KOD-plus DNA polymerase in a final volume
of 50 μl. PCR consisted of 28 cycles with denaturation at 94 °C
for 15 s, annealing at 58 °C for 30 s and extension at 68 °C for
1
1
50 s. Initial denaturation and final extension were at 94 °C for
20 s and at 68 °C for 420 s, respectively. The resulting PCR
product was isolated by electrophoresis on a 1.0% TAE agarose
gel and gel-purified with a MinElute Gel Extraction kit, and A-
tailing of the 3′-flanking region was performed using Taq DNA
polymerase. The PCR product was directly introduced into
pGEM-T vector using the TA cloning system. The plasmid was
sequenced in both forward and reverse directions using a
BigDye terminator cycle sequencing reaction kit v3.1 to
confirm that there were no PCR errors. DNA fragment
corresponding to OR was cut from the pGEM-T plasmid with
HindIII and KpnI, and subsequently subcloned into the
pYES3/CT expression vector digested with HindIII and KpnI
(
pYES3/OR). The expression plasmid was sequenced to verify
the correct orientation with respect to the promoter for pYES3/
CT. Saccharomyces cerevisiae INVSc1 strain was transformed
with the plasmid pYES3/OR by the lithium acetate procedure
(
Schiestl and Gietz, 1989), and the transformant (INVSc1/OR)
was selected using TRP1 auxotrophic marker.
Expression of humCYP2E1 and monCYP2E1 enzymes
The full-length cDNA encoding humCYP2E1 was amplified
by PCR from humCYP2E1 cDNA cloned into pENTR/D-
TOPO vector (Hanioka et al., 2003) as a template using
humCYP2E1-F and humCYP2E1-R primer pair. The PCR
reaction mix contained 1×PCR buffer, 0.2 mM dNTPs, each
primer at 0.5 μM and 1 U KOD-plus DNA polymerase in a final
volume of 50 μl. PCR consisted of 28 cycles with denaturation
at 94 °C for 15 s, annealing at 60 °C for 30 s and extension at
The transformant INVSc1/OR was transformed with the
expression plasmid containing humCYP2E1 or monCYP2E1
by the lithium acetate procedure (Schiestl and Gietz, 1989).
TRP1 and URA3 auxotrophic markers were used to select
these transformants. For the production of CYP2E1 proteins,
yeast cells were grown at 30 °C with shaking to an OD600 of
4.0 in 200 ml of selective medium containing 2% glucose as the
carbon source. Cells corresponding to 0.4 OD600 were
6
8 °C for 90 s. Initial denaturation and final extension were at

N. Hanioka et al. / Life Sciences 81 (2007) 1436–1445
1439
inoculated into 2000 ml of induction medium containing 2%
galactose and 1% raffinose as the carbon source. After
cultivation at 30 °C for 24 h, microsomal fractions from yeast
cells were prepared by the method of Oeda et al. (1985) with
some modifications. The cells were harvested by centrifugation
at 5000 g for 10 min at 4 °C, washed with 400 ml of water and
centrifuged at 5000 g for 10 min at 4 °C. The pellets were
suspended in 200 ml of buffer A (20 mM Tris–HCl (pH 7.4)
containing 2 M sorbitol, 0.1 mM dithiothreitol, 0.1 mM EDTA
and 50 U/ml Zymolyase 100T), and incubated at 35 °C for 2 h
with gentle shaking. The pellets obtained after centrifugation at
5000 g for 20 min at 4 °C were washed again with 200 ml of
buffer A before being resuspended in 100 ml of buffer B
(20 mM Tris–HCl (pH 7.4) containing 0.6 M sorbitol, 0.1 mM
dithiothreitol, 0.1 mM EDTA and 0.5 mM phenylmethylsulfo-
nyl fluoride), and sonicated on ice 10 times (30 s each time). For
Fig. 1. Nucleotide and deduced amino acid sequences of monCYP2E1. The sequences identified in this study are in bold.

1440
N. Hanioka et al. / Life Sciences 81 (2007) 1436–1445
comparison, INVSc1 transformants with pYES2/CT as a null
vector were used as a negative control. The cell lysates were
centrifuged at 9000 g for 20 min at 4 °C, and the supernatants
were further centrifuged at 105,000 g for 60 min at 4 °C. The
resulting microsomal pellets were resuspended in 50 mM
potassium phosphate buffer (pH 7.4) containing 20% glycerol
and stored at −80 °C until use.
as described by Towbin et al. (1979). The sheet was incubated
with rabbit anti-human CYP2E1 antibody (diluted at 1:10,000)
as the primary antibody and then with peroxidase-conjugated
goat anti-rabbit immunoglobulin (diluted at 1:5000) as the
secondary antibody. Immunoreactive proteins were visualized
with chemifluorescence (enhanced chemiluminescence-plus
reagents), and the band densities were relatively determined
with Scion Image v4.0 (Scion Corporation, Frederick, MD).
Assay for CYP2E1 holo- and apoproteins
Assay for CYP2E1-dependent enzymatic activities
Microsomal fractions were diluted to a protein concentration
of 1.0 mg/ml (liver microsomes) or 5.0 mg/ml (yeast cell
microsomes expressing CYP2E1s) with 100 mM potassium
phosphate buffer (pH 7.4) containing 20% (v/v) glycerol and
Chlorzoxazone 6-hydroxylation and 4-nitrophenol 2-hydrox-
ylation activities were determined by high-performance liquid
chromatography (HPLC) as described previously with some
modifications (Lucas et al., 1993; Tassaneeyakul et al., 1993b).
The incubation mixture contained chlorzoxazone (50–
5000 μM) or 4-nitrophenol (50–5000 μM) as a substrate, liver
microsomes (200 μg protein/ml) or yeast cell microsomes
expressing CYP2E1s (500 μg protein/ml) and an NADPH-
0
.4% (w/v) Emulgen 911, and total functional CYP contents
were spectrophotometrically measured as reduced carbon
monoxide (CO) spectra according to the method of Omura
−
1
−1
and Sato (1964) using 91 mM
cm
as an absorption
coefficient for the 450–490 wavelength couple. Total CYP2E1
protein levels of holo- and apoforms in yeast cell microsomes
were determined by Western blot analysis. Microsomal
fractions (5.0 μg protein) were separated by 10% sodium
dodecyl sulfate–polyacrylamide gel electrophoresis (Laemmli,
+
generating system (1 mM NADP , 10 mM glucose 6-phosphate,
2 U/ml glucose 6-phosphate dehydrogenase and 5 mM MgCl2)
in 50 mM potassium phosphate buffer (pH 7.4) in a final volume
of 500 μl. Each substrate was dissolved in methanol/dimethyl
sulfoxide (50:50, v/v). The final concentration of the organic
1
970) and electrotransferred to a polyvinylidene fluoride sheet
Fig. 2. Deduced amino acid sequence alignment of humCYP2E1 and monCYP2E1. The asterisks indicate the same amino acid residues between humCYP2E1 and
monCYP2E1. SRSs are shown in shaded boxes.

N. Hanioka et al. / Life Sciences 81 (2007) 1436–1445
1441
solvent (methanol and/or dimethyl sulfoxide) in the incubation
Data analysis
mixture was 1% (v/v). The reaction was initiated by the addition
of microsomes from livers or yeast cells expressing CYP2E1
after preincubation at 37 °C for 1 min. After incubation at 37 °C
for 20 min, the reaction was terminated by the addition of 4 ml of
ethyl acetate. Samples for chlorzoxazone 6-hydroxylation and 4-
nitrophenol 2-hydroxylation activities were spiked with internal
standards (2 nmol of 4-nitrophenol and 5 nmol of salicylamide,
respectively) and vigorously vortexed for 2 min. After
centrifugation at 2000 g for 15 min, the organic phase was
evaporated to dryness under a gentle stream of nitrogen at 35 °C.
The residues were dissolved in 200 μl of methanol–water
Kinetic parameters such as K and V
for chlorzoxazone
m
max
6-hydroxylation and 4-nitrophenol 2-hydroxylation were esti-
mated by analyzing Michaelis–Menten plots using Prism v4.0
software (GraphPad Software, San Diego, CA). Intrinsic
clearance values were determined as the ratio of Vmax /K . All
m
values are expressed as the mean±S.D. of three donors/animals
or three separate experiments derived from independent
preparations. Statistical comparisons were made with Student's
t-test, and differences were considered statistically significant
when the p value was b0.05.
(
50:50, v/v) and analyzed by HPLC.
The HPLC system consisted of an AS-8021 autosampler
Tosoh, Tokyo, Japan), a DP-8020 pump (Tosoh), a CO-8020
Results
(
column oven (Tosoh), a UV-8020 UV detector (Tosoh) and a
GT-102 degasser (Lab-Quatec, Tokyo, Japan) equipped with an
Inertsil ODS-80A column (4.6 mm i.d.×150 mm; GL Sciences,
Tokyo, Japan). The column was maintained at 40 °C. Data
acquisition was accomplished using LC-8020 v1.3 software
Sequence analysis
Sequence analysis determined that humCYP2E1 and
monCYP2E1 cDNAs have an open reading frame of 1484 bp,
and cDNAs encode the respective proteins of 493 amino acids.
Fig. 1 shows the nucleotide and deduced amino acid sequences
of monCYP2E1. As shown in Fig. 2, there are 28 different
(
Tosoh). The product (6-hydroxychlorzoxazone) in the assay for
chlorzoxazone 6-hydroxylation activity was eluted isocratically
with 80 mM acetic acid–acetonitrile (82:18, v/v) at a flow rate
of 1.0 ml/min. UV detection was performed at 295 nm.
Retention times of 6-hydroxychlorzoxazone, chlorzoxazone
and 4-nitrophenol were 5.6, 26.6 and 15.8 min, respectively.
The limit of quantification for 6-hydroxychlorzoxazone was
1
0 pmol/ml. The 6-hydroxychlorzoxazone formation was linear
for at least 30 min in microsomes from livers and yeast cells
expressing CYP2E1s of humans and cynomolgus monkeys
(
200 μg/ml for liver microsomes and 500 μg/ml for yeast cell
microsomes expressing CYP2E1s). In the assay for 4-
nitrophenol 2-hydroxylation activity, the product (4-nitrocate-
chol) was eluted isocratically with 20 mM triethylamine–
acetonitrile–methanol (82:12:6, v/v/v) at a flow rate of 1.0 ml/
min. The pH of the aqueous portion was adjusted to 3.0 with
phosphoric acid. UV absorption was monitored at 250 nm.
Retention times of 4-nitrocatechol, 4-nitrophenol and salicyla-
mide were 11.9, 22.6 and 9.1 min, respectively. The limit of
quantification for 4-nitrocatechol was 20 pmol/ml. The 4-
nitrocatechol formation was linear for at least 30 min in
microsomes from livers and yeast cells expressing CYP2E1s of
humans and cynomolgus monkeys (200 μg/ml for liver
microsomes and 500 μg/ml for yeast cell microsomes expres-
sing CYP2E1s). Standard curve samples for both assays were
prepared in the same manner as the incubation samples. Intra-
day (n=5) and inter-day (n=5) precision did not exceed 10% in
any of the assays.
Other methods
Fig. 3. Reduced CO-difference spectra of microsomes from livers and yeast cells
expressing CYP2E1s of humans and cynomolgus monkeys. (A) Liver
microsomes. (B) Yeast cell microsomes expressing CYP2E1s. Typical results
of pooled microsomes from three independent preparations. The microsomal
protein concentrations used were 1.0 mg/ml for liver microsomes and 5.0 mg/ml
for yeast cell microsomes expressing CYP2E1s. Broken line, human liver
microsomes or humCYP2E1; solid line, cynomolgus monkey liver microsomes
or monCYP2E1.
Protein concentrations of microsomes from livers and yeast
cells expressing CYP2E1s of humans and cynomolgus
monkeys were determined by the method of Lowry et al.
(
1951) using bovine serum albumin as a standard. OR activities
were measured as described previously, with a substrate
concentration of 50 μM (Phillips and Langdon, 1962).

1442
N. Hanioka et al. / Life Sciences 81 (2007) 1436–1445
Table 2
Table 3
CYP contents and OR activities in microsomes from livers and yeast cells
Kinetic parameters for chlorzoxazone 6-hydroxylation by microsomes from
expressing CYP2E1s of humans and cynomolgus monkeys
livers and yeast cells expressing CYP2E1s of humans and cynomolgus monkeys
c
a
Enzyme
CYP
OR
Enzyme
K
m
V
max
V
max /K
m
a
b
b
c
d
e
Reduced CO
WB
Protein
CYP
Protein
CYP
HLM
MLM
humCYP2E1
monCYP2E1
316±128
563±105
38.4±19.4
16.4±2.7
100±19.7
74.1±11.0
100±13.4
69.4±16.0
209±48
145±22
233±98
288±88
HLM
MLM
822±322 909±130
1250±560
1270±50
272±61
⁎
⁎
⁎
418±42
532±56
170±37
humCYP2E1 627±19
monCYP2E1 708±115 101±10
4.88±1.34
6.29±1.67
7.78±2.13
8.81±0.98
⁎
143±14
Each value represents the mean±S.D. of three separate experiments derived
from independent preparations. HLM, human liver microsomes; MLM,
cynomolgus monkey liver microsomes.
Each value represents the mean±S.D. of three separate experiments derived
from independent preparations. HLM, human liver microsomes; MLM,
cynomolgus monkey liver microsomes.
a
⁎
⁎⁎
Reduced CO-difference spectral analysis (pmol/mg protein).
Western blot analysis (% of HLM or humCYP2E1).
nmol/min/mg protein.
pb0.05, pb0.01 compared with HLM or humCYP2E1.
b
a
μM.
c
b
pmol/min/mg protein.
pmol/min/pmol CYP.
nl/min/mg protein.
nl/min/pmol CYP.
c
d
e
amino acid residues between humCYP2E1 and monCYP2E1,
with homology of 94.3% in the amino acid sequence.
summation of upper and lower bands) were human liver
microsomes (100) and cynomolgus monkey liver microsomes
Expression of humCYP2E1 and monCYP2E1 enzymes
(74.1), and humCYP2E1 (100) and monCYP2E1 (69.4), where
CYP contents of microsomes from livers and yeast cells
expressing CYP2E1s of humans and cynomolgus monkeys
were examined by reduced CO-difference spectral and Western
blot analyses. The reduced CO-difference spectra of yeast cell
microsomes expressing humCYP2E1 and monCYP2E1 as well
as human and cynomolgus monkey liver microsomes showed a
Soret peak at around 450 nm (Fig. 3). CYP contents of enzyme
sources in humans were 316 pmol/mg protein for liver
microsomes and 38.4 pmol/mg protein for yeast cell micro-
somes expressing humCYP2E1. The levels in microsomes from
livers and yeast cells expressing monCYP2E1 in cynomolgus
monkeys were 1.78-fold and 42.7% of those in humans,
respectively, although no significant differences were statisti-
cally observed (Table 2). The expression levels of CYP2E1
proteins in liver and yeast cell microsomes of humans and
cynomolgus monkeys were also assessed by Western blot
analysis which recognized both holo- and apoforms (Fig. 4). All
enzyme sources except the negative control (mock) yielded
immunodetectable CYP2E1 protein; however, the staining band
intensities of microsomes from cynomolgus monkey livers and
yeast cells expressing monCYP2E1 were weak compared with
those of enzyme sources in humans. The relative levels (the
human values were normalized to 100 (Table 2).
OR activities in microsomes from livers and yeast cells
expressing CYP2E1s of humans and cynomolgus monkeys
were also determined. OR activities of enzyme sources in
humans were 209 nmol/min/mg protein for liver microsomes
and 233 nmol/min/mg protein for yeast cell microsomes
expressing humCYP2E1. The activities in microsomes from
livers and yeast cells expressing monCYP2E1 in cynomolgus
monkeys were comparable to those in humans; there were no
significant differences in the OR activities of humans and
cynomolgus monkeys in any enzyme source (Table 2).
Enzymatic properties of humCYP2E1 and monCYP2E1
To obtain information on the enzymatic properties of human and
cynomolgus monkey CYP2E1s, kinetic analyses for chlorzox-
azone 6-hydroxylation and 4-nitrophenol 2-hydroxylation by yeast
cell microsomes expressing humCYP2E1 and monCYP2E1 as
Table 4
Kinetic parameters for 4-nitrophenol 2-hydroxylation by microsomes from
livers and yeast cells expressing CYP2E1s of humans and cynomolgus monkeys
a
Enzyme
K
m
V
max
V
max /K
m
b
c
d
e
Protein
422±133 1250±570
434±58 646±142
CYP
Protein
CYP
HLM
MLM
2990±1090
1480±210
humCYP2E1 514±134 139±31
3.98±1.11 277±64
8.41±4.47
5.46±0.51
⁎
⁎⁎
monCYP2E1 736±66
65.2±2.8 4.04±0.71 88.8±6.0
Each value represents the mean±S.D. of three separate experiments derived
from independent preparations. HLM, human liver microsomes; MLM,
cynomolgus monkey liver microsomes.
⁎
⁎⁎
Fig. 4. Immunoblotting of microsomes from livers and yeast cells expressing
CYP2E1s of humans and cynomolgus monkeys. Typical results of pooled
microsomes from three independent preparations. The microsomal protein levels
applied were 5.0 μg/lane for both microsomes from livers and yeast cells
expressing CYP2E1s. HLM, human liver microsomes; MLM, cynomolgus
monkey liver microsomes.
pb0.05, pb0.01 compared with HLM or humCYP2E1.
a
μM.
b
pmol/min/mg protein.
pmol/min/pmol CYP.
nl/min/mg protein.
nl/min/pmol CYP.
c
d
e

N. Hanioka et al. / Life Sciences 81 (2007) 1436–1445
1443
well as by liver microsomes from humans and cynomolgus
monkeys were performed. No activity in yeast cell microsomes of
the negative control was detected with any substrate (data not
shown). The calculated kinetic parameters estimated by Michaelis–
animal model for the development of medicines, and particularly
for the characterization of pharmacokinetics and toxicological
properties of novel molecules, little is known about the
enzymatic properties of monkey CYP enzymes. The functional
evaluation of human and cynomolgus monkey CYP2E1
enzymes should provide important information for the predic-
tion and extrapolation of xenobiotic metabolism. Enzymatic
activities in microsomes from yeast cells expressing human and
cynomolgus monkey CYP2E1s were therefore investigated by
kinetic analysis. To this end, we used chlorzoxazone 6-
hydroxylation and 4-nitrophenol 2-hydroxylation as probes for
CYP2E1.
Menten plots are summarized in Tables 3 and 4. The K values of
m
human and cynomolgus monkey liver microsomes for chlorzox-
azone 6-hydroxylation were 822 and 418 μM, respectively. The Km
values of yeast cell microsomes expressing humCYP2E1 and
monCYP2E1 were 627 and 708 μM, respectively. There were no
significant differences in the Km values of liver and yeast cell
microsomes between humans and cynomolgus monkeys. The Vmax
and Vmax /K values of enzyme sources in humans on the basis of
m
the microsomal protein level were 909 pmol/min/mg protein and
The expression of CYP2E1 protein was confirmed by
Western blot analysis. Anti-human CYP2E1 antibody cross-
reacted with two proteins in microsomes from livers and yeast
cells expressing CYP2E1s of humans and cynomolgus
monkeys, and the staining intensities of both bands of cyno-
molgus monkeys were slightly weaker than those of humans.
Multiple CYP2E1-like bands have been previously detected in
ethanol-induced rat livers and phenobarbital-induced monkey
brains (Neve and Ingelman-Sundberg, 1999; Lee et al., 2006).
The profile of detected protein bands was also different between
microsomes from livers and yeast cells expressing CYP2E1s.
The results obtained in this study agreed with those of previous
reports, and this phenomenon may be due the alteration of
posttranslation modifications or cellular targeting. Furthermore,
450-nm absorbance was measured by reduced CO-difference
spectra in yeast cell microsomes as well as in liver microsomes.
The functional CYP level of monCYP2E1 was lower than that
of humCYP2E1, although no significance was observed. Thus,
the expression of functional CYP2E1 proteins in yeast cell
microsomes expressing humCYP2E1 and monCYP2E1 as well
as in human and cynomolgus monkey liver microsomes was
confirmed.
Chlorzoxazone and 4-nitrophenol have been suggested to be
typical substrates for CYP2E1 (Rendic and Di Carlo, 1997).
Enzymatic activities in this study were analyzed in two ways:
as pmol/min/mg protein (on the basis of microsomal protein
level), and as pmol/min/pmol CYP (on the basis of CYP
holoprotein level). Both yeast cell microsomes expressing
humCYP2E1 and monCYP2E1 were capable of oxidizing
chlorzoxazone and 4-nitrophenol as well as human and cyno-
molgus monkey liver microsomes at the substrate concentra-
tions examined. We confirmed that the kinetics for these
reactions in microsomes from liver and yeast cells expressing
CYP2E1s of humans and cynomolgus monkeys fits the
Michaelis–Menten equation.
1250 nl/min/mg protein for liver microsomes, and 170 pmol/min/
mg protein and 272 nl/min/mg protein for yeast cell microsomes
expressing humCYP2E1, respectively. The Vmax values of
cynomolgus monkeys were significantly lower than those of
humans (58.5–59.4%), although the Vmax /K value of cynomol-
m
gus monkey liver microsomes was comparable to that of human
liver microsomes. When the activities were normalized to CYP
holoprotein levels to assess the intrinsic function of CYP2E1
enzymes, the Vmax and Vmax /Km values of humCYP2E1 were
4.88 pmol/min/pmol CYP and 7.78 nl/min/pmol CYP, respectively.
No significant differences in Vmax and Vmax /K values on the basis
m
of CYP holoprotein level were observed between humCYP2E1
and monCYP2E1.
The Km values of human and cynomolgus monkey liver
microsomes for 4-nitrophenol 2-hydroxylation were 422 and
4
34 μM, respectively. The K values of yeast cell microsomes
m
expressing CYP2E1s were 514 μM for humCYP2E1 and
36 μM for monCYP2E1, and there were no significant
7
differences in the Km values of humans and cynomolgus
monkeys in any enzyme source. Vmax and Vmax /Km values of
enzyme sources in humans on the basis of microsomal protein
level were 1250 pmol/min/mg protein and 2990 nl/min/mg
protein for liver microsomes, and 139 pmol/min/mg protein and
2
77 nl/min/mg protein for yeast cell microsomes expressing
humCYP2E1, respectively. The levels of cynomolgus monkeys
were 32.1–51.7% of those in humans. Vmax and Vmax /Km
values of yeast cell microsomes expressing CYP2E1s on the
basis of CYP holoprotein level were 3.98 pmol/min/pmol
CYP and 8.41 nl/min/pmol CYP for humCYP2E1, and
4
.04 pmol/min/pmol CYP and 5.46 nl/min/pmol CYP for
humCYP2E1. Vmax and Vmax /Km values of monCYP2E1 on
the basis of CYP holoprotein level were comparable to those
of humCYP2E1.
Discussion
K values for CYP2E1-dependent reactions of liver micro-
m
somes in previous studies have been estimated as follows:
chlorzoxazone 6-hydroxylation, 39 μM for humans and 77 μM
for cynomolgus monkeys; 4-nitrophenol 2-hydroxylation,
30 μM for humans and 14 μM for cynomolgus monkeys
(Peter et al., 1991; Tassaneeyakul et al., 1993a; Amato et al.,
CYP2E1, a member of the CYP superfamily, toxicologically
plays a key role in the oxidative metabolism of various drugs,
precarcinogens and solvents (Gonzalez, 1990; Guengerich et al.,
1
991). CYP2E1 activity and expression have also been shown to
be influenced by genetic, physiological and environmental
factors (Gonzalez, 1990; Kim and O'Shea, 1995; Lucas et al.,
1998). K values in this study (chlorzoxazone 6-hydroxylation,
m
820 μM for humans and 420 μM for cynomolgus monkeys; 4-
nitrophenol 2-hydroxylation, 420 μM for humans and 430 μM
for cynomolgus monkeys) were higher than those previously
1
995, 1998; McCarver et al., 1998). Although monkeys,
including cynomolgus monkeys, are commonly used as an

1444
N. Hanioka et al. / Life Sciences 81 (2007) 1436–1445
reported in both species, although the cause of this difference is
unclear at present. With respect to recombinant CYP2E1
enzymes, Km values of human CYP2E1 enzyme expressed in
heterologous cells such as T. ni, COS-1, COS-7 and human B-
lymphoblastoid cells have been reported to be 81–660 μM for
chlorzoxazone 6-hydroxylation and 21–141 μM for 4-nitro-
phenol 2-hydroxylation (Tassaneeyakul et al., 1993a; Fair-
brother et al., 1998; Shimada et al., 1999; Hanioka et al., 2003).
substrates. Further studies are required to elucidate the
relationship between the protein structure and substrate
specificity of primate CYP2E1s.
In conclusion, we expressed human and cynomolgus monkey
CYP2E1 enzymes in yeast cells, and showed chlorzoxazone 6-
hydroxylation and 4-nitrophenol 2-hydroxylation activities in
yeast cell microsomes expressing CYP2E1s as well as in liver
microsomes of humans and cynomolgus monkeys. The functional
CYP levels of monCYP2E1 expressed in yeast cell microsomes
were lower than those of humCYP2E1 in both reduced CO-
difference spectral and Western blot analyses, although no
significant differences were statistically observed. There were
no significant differences in Km values for chlorzoxazone 6-
hydroxylation and 4-nitrophenol 2-hydroxylation between
humans and cynomolgus monkeys in both microsomes from
livers and yeast cells expressing CYP2E1s. Vmax and Vmax/Km
values of monCYP2E1 on the basis of microsomal protein level
were significantly lower than those of humCYP2E1 in both
reactions, whereas the kinetic parameters of humCYP2E1 and
monCYP2E1 on the basis of CYP holoprotein level exhibited
similar values. These findings suggest that human and cynomol-
gus monkey CYP2E1 enzymes have high homology in their
amino acid sequences, and that their enzymatic properties are
considerably similar. The information gained in this study should
help with in vivo extrapolation and to assess the toxicity of
xenobiotics.
Thus, K values of human CYP2E1 enzyme for chlorzoxazone
m
6
-hydroxylation and 4-nitrophenol 2-hydroxylation varied
among heterologous cell expression systems, and Km values
of human and cynomolgus monkey CYP2E1 enzymes
expressed in yeast cells were first estimated in this study.
Additionally, there were no significant differences in K values
m
of liver microsomes for chlorzoxazone 6-hydroxylation and 4-
nitrophenol 2-hydroxylation between humans and cynomolgus
monkeys. Km values of yeast cell microsomes expressing
humCYP2E1 and monCYP2E1 were comparable to those of
human and cynomolgus monkey liver microsomes, respective-
ly, suggesting that CYP2E1 is the predominant isoform
responsible for oxidation in both species.
There were no notable differences in Vmax and Vmax /Km
values of liver microsomes for chlorzoxazone 6-hydroxylation
and 4-nitrophenol 2-hydroxylation between humans and
cynomolgus monkeys. In yeast cell microsomes expressing
CYP2E1s, Vmax and Vmax /Km values of monCYP2E1 on the
basis of the microsomal protein level were significantly lower
than those of humCYP2E1 in both reactions, whereas kinetic
parameters on the basis of CYP holoprotein level exhibited
similar values between humCYP2E1 and monCYP2E1. The
low Vmax values of monCYP2E1 on the basis of microsomal
protein compared with those of humCYP2E1 are suggested to
be attributed to differences in CYP2E1 holoprotein levels but
not in the intrinsic enzyme function.
Acknowledgments
This work was supported in part by Grants-in-Aid for
Scientific Research (17390035 and 18590116) from the
Ministry of Education, Culture, Sports, Science and Technology
of Japan.
X-ray crystal structures of several mammalian CYP enzymes
rabbit CYP2B4 and CYP2C5, and human CYP2C8, CYP2C9,
References
(
CYP2D6 and CYP3A4) have been reported to date (Williams
et al., 2000, 2003, 2004; Schoch et al., 2004; Scott et al., 2004;
Rowland et al., 2006). Homology modeling for human CYP2E1
has been generated using the CYP2C5 crystal structure as a
template, and six substrate recognition sites (SRSs) and a
putative active site for CYP2E1 have been identified (Lewis,
Amato, G., Longo, V., Mazzaccaro, A., Gervasi, P.G., 1998. Chlorzoxazone
6
-hydroxylase and p-nitrophenol hydroxylase as the most suitable
activities for assaying cytochrome P450 2E1 in cynomolgus monkey
liver. Drug Metabolism and Disposition 26 (5), 483–489.
Fairbrother, K.S., Grove, J., de Waziers, I., Steimel, D.T., Day, C.P., Crespi, C.L.,
Daly, A.K., 1998. Detection and characterization of novel polymorphisms in
the CYP2E1 gene. Pharmacogenetics 8 (6), 543–552.
Gonzalez, F.J., 1990. Molecular genetics of the P-450 superfamily. Pharmacol-
ogy and Therapeutics 45 (1), 1–38.
Guengerich, F.P., Kim, D.H., Iwasaki, M., 1991. Role of human cytochrome P-450
IIE1 in the oxidation of many low molecular weight cancer suspects. Chemical
Research in Toxicology 4 (2), 168–179.
Hanioka, N., Tanaka-Kagawa, T., Miyata, Y., Matsushima, E., Makino, Y.,
Ohno, A., Yoda, R., Jinno, H., Ando, M., 2003. Functional characterization
of three human cytochrome P450 2E1 variants with amino acid substitu-
tions. Xenobiotica 33 (6), 575–586.
2
002, 2003; Lewis et al., 2003). The distance between oxidation
sites on substrate molecules (6-position for chlorzoxazone; 2-
position for 4-nitrophenol) and haem iron was 5.7 Å. Phe205
and Phe298 of CYP2E1 are able to form π–π stacking with the
benzoxazone ring of chlorzoxazone or benzene ring of 4-
nitrophenol. Asp202 and Thr303 of CYP2E1 in chlorzoxazone
6
-hydroxylation enter into hydrogen bonding with hydroxyl and
chloro groups of the substrate, respectively. With respect to 4-
nitrophenol 2-hydroxylation, Asp202 and Thr303 residues
serve to anchor the 4-nitrophenol molecule via nitro and
hydroxyl groups, respectively. These amino acid residues were
in SRS-2 or SRS-4, and were identical between humCYP2E1
and monCYP2E1. Accordingly, it is considered that the
functional similarities of humCYP2E1 and monCYP2E1 are
due to the identity of amino acid resides interacting with
Ingelman-Sundberg, M., Johansson, I., Yin, H., Terelius, Y., Eliasson, E., Clot,
P., Albano, E., 1993. Ethanol-inducible cytochrome P4502E1: genetic
polymorphism, regulation, and possible role in the etiology of alcohol-
induced liver disease. Alcohol 10 (6), 447–452.
Kim, R.B., O'Shea, D., 1995. Interindividual variability of chlorzoxazone 6-
hydroxylation in men and women and its relationship to CYP2E1 genetic
polymorphisms. Clinical Pharmacology and Therapeutics 57 (6), 645–655.
Komori, M., Kikuchi, O., Sakuma, T., Funaki, J., Kitada, M., Kamataki, T.,
1992. Molecular cloning of monkey liver cytochrome P-450 cDNAs:

N. Hanioka et al. / Life Sciences 81 (2007) 1436–1445
1445
similarity of the primary sequences to human cytochromes P-450.
Biochimica et Biophysica Acta 1171 (2), 141–146.
Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the
head of bacteriophage T4. Nature 227 (5259), 680–685.
Lee, A.M., Joshi, M., Yue, J., Tyndale, R.F., 2006. Phenobarbital induces
monkey brain CYP2E1 protein but not hepatic CYP2E1, in vitro or in vivo
chlorzoxazone metabolism. European Journal of Pharmacology 552 (1-3),
Rendic, S., Di Carlo, F.J., 1997. Human cytochrome P450 enzymes: a status
report summarizing their reactions, substrates, inducers, and inhibitors. Drug
Metabolism Reviews 29 (1-2), 413–580.
Romanos, M.A., Scorer, C.A., Clare, J.J., 1992. Foreign gene expression in
yeast: a review. Yeast 8 (6), 423–488.
Rowland, P., Blaney, F.E., Smyth, M.G., Jones, J.J., Leydon, V.R., Oxbrow, A.K.,
Lewis, C.J., Tennant, M.G., Modi, S., Eggleston, D.S., Chenery, R.J., Bridges,
A.M., 2006. Crystal structure of human cytochrome P450 2D6. Journal of
Biological Chemistry 281 (11), 7614–7622.
Schiestl, R.H., Gietz, R.D., 1989. High efficiency transformation of intact yeast
cells using single stranded nucleic acids as a carrier. Current Genetics 16 (5-6),
339–346.
Schoch, G.A., Yano, J.K., Wester, M.R., Griffin, K.J., Stout, C.D., Johnson, E.F.,
2004. Structure of human microsomal cytochrome P450 2C8: evidence for a
peripheral fatty acid binding site. Journal of Biological Chemistry 279 (10),
9497–9503.
Scott, E.E., White, M.A., He, Y.A., Johnson, E.F., Stout, C.D., Halpert, J.R.,
2004. Structure of mammalian cytochrome P450 2B4 complexed with 4-
(4-chlorophenyl)imidazole at 1.9-Å resolution: insight into the range of
P450 conformations and the coordination of redox partner binding. Journal
of Biological Chemistry 279 (26), 27294–27301.
151–158.
Lewis, D.F.V., 2002. Homology modelling of human CYP2 family enzymes
based on the CYP2C5 crystal structure. Xenobiotica 32 (4), 305–323.
Lewis, D.F.V., 2003. Essential requirements for substrate binding affinity and
selectivity toward human CYP2 family enzymes. Archives of Biochemistry
and Biophysics 409 (1), 32–44.
Lewis, D.F.V., Lake, B.G., Bird, M.G., Loizou, G.D., Dickins, M., Goldfarb, P.S.,
2003. Homology modelling of human CYP2E1 based on the CYP2C5 crystal
structure: investigation of enzyme-substrate and enzyme-inhibitor interactions.
Toxicology in Vitro 17 (1), 93–105.
Lieber, C.S., 1997. Cytochrome P-4502E1: its physiological and pathological
role. Physiological Reviews 77 (2), 517–544.
Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein
measurement with the Folin phenol reagent. Journal of Biological Chemistry
1
93 (1), 265–275.
Shimada, T., Yamazaki, H., Mimura, M., Inui, Y., Guengerich, F.P., 1994.
Interindividual variations in human liver cytochrome P-450 enzymes
involved in the oxidation of drugs, carcinogens and toxic chemicals: studies
with liver microsomes of 30 Japanese and 30 Caucasians. The Journal of
Pharmacology and Experimental Therapeutics 270 (1), 414–423.
Shimada, T., Tsumura, F., Yamazaki, H., 1999. Prediction of human liver
microsomal oxidations of 7-ethoxycoumarin and chlorzoxazone with kinetic
parameters of recombinant cytochrome P-450 enzymes. Drug Metabolism
and Disposition 27 (11), 1274–1280.
Lucas, D., Berthou, F., Girre, C., Poitrenaud, F., Menez, J.F., 1993. High-
performance liquid chromatographic determination of chlorzoxazone and
6
-hydroxychlorzoxazone in serum: a tool for indirect evaluation of
cytochrome P450 2E1 activity in humans. Journal of Chromatography 622
1), 79–86.
(
Lucas, D., Farez, C., Bardou, L.G., Vaisse, J., Attali, J.R., Valensi, P., 1998.
Cytochrome P450 2E1 activity in diabetic and obese patients as assessed by
chlorzoxazone hydroxylation. Fundamental and Clinical Pharmacology 12
(
5), 553–558.
Tassaneeyakul, W., Veronese, M.E., Birkett, D.J., Gonzalez, F.J., Miners, J.O.,
1993a. Validation of 4-nitrophenol as an in vitro substrate probe for human
liver CYP2E1 using cDNA expression and microsomal kinetic techniques.
Biochemical Pharmacology 46 (11), 1975–1981.
Tassaneeyakul, W., Veronese, M.E., Birkett, D.J., Miners, J.O., 1993b. High-
performance liquid chromatographic assay for 4-nitrophenol hydroxylation,
a putative cytochrome P-4502E1 activity, in human liver microsomes.
Journal of Chromatography 616 (1), 73–78.
Towbin, H., Staehelin, T., Gordon, J., 1979. Electrophoretic transfer of proteins
from polyacrylamide gels to nitrocellulose sheets: procedure and some
applications. Proceedings of the National Academy of Sciences of the
United States of America 76 (9), 4350–4354.
Umeno, M., McBride, O.W., Yang, C.S., Gelboin, H.V., Gonzalez, F.J., 1988.
Human ethanol-inducible P450IIE1: complete gene sequence, promoter
characterization, chromosome mapping, and cDNA-directed expression.
Biochemistry 27 (25), 9006–9013.
Lucas, D., Menez, C., Girre, C., Berthou, F., Bodenez, P., Joannet, I., Hispard,
E., Bardou, L.G., Menez, J.F., 1995. Cytochrome P450 2E1 genotype and
chlorzoxazone metabolism in healthy and alcoholic Caucasian subjects.
Pharmacogenetics 5 (5), 298–304.
McCarver, D.G., Byun, R., Hines, R.N., Hichme, M., Wegenek, W., 1998. A
genetic polymorphism in the regulatory sequences of human CYP2E1:
association with increased chlorzoxazone hydroxylation in the presence of
obesity and ethanol intake. Toxicology and Applied Pharmacology 152 (1),
2
76–281.
Narimatsu, S., Takatsu, N., Yamano, S., Inoue, Y., Hanioka, N., Kiryu, K.,
Naito, S., Gonzalez, F.J., Yamamoto, S., 2006. The effect of dimethyl
sulfoxide on the function of cytochrome P450 2D6 in HepG2 cells upon the
co-expression with NADPH-cytochrome P450 reductase. Chemico-Biolog-
ical Interactions 159 (1), 47–57.
Nelson, D.R., Koymans, L., Kamataki, T., Stegeman, J.J., Feyereisen, R.,
Waxman, D.J., Waterman, M.R., Gotoh, O., Coon, M.J., Estabrook, R.W.,
Gunsalus, I.C., Nebert, D.W., 1996. P450 superfamily: update on new
sequences, gene mapping, accession numbers and nomenclature. Pharma-
cogenetics 6 (1), 1–42.
Williams, P.A., Cosme, J., Sridhar, V., Johnson, E.F., McRee, D.E., 2000.
Mammalian microsomal cytochrome P450 monooxygenase: structural
adaptations for membrane binding and functional diversity. Molecular Cell
5 (1), 121–131.
Neve, E.P., Ingelman-Sundberg, M., 1999. A soluble NH
2
-terminally truncated
Williams, P.A., Cosme, J., Ward, A., Angove, H.C., Vinković, D.M., Jhoti, H.,
2003. Crystal structure of human cytochrome P450 2C9 with bound
warfarin. Nature 424 (6947), 464–468.
catalytically active form of rat cytochrome P450 2E1 targeted to liver
mitochondria. FEBS Letters 460 (2), 309–314.
Oeda, K., Sakaki, T., Ohkawa, H., 1985. Expression of rat liver cytochrome
P-450MC cDNA in Saccharomyces cerevisiae. DNA 4 (3), 203–210.
Omura, T., Sato, R., 1964. The carbon monoxide-binding pigment of liver
microsomes. I. Evidence for its hemoprotein nature. Journal of Biological
Chemistry, 239 (7), 2370–2378.
Perrot, N., Nalpas, B., Yang, C.S., Beaune, P.H., 1989. Modulation of
cytochrome P450 isozymes in human liver, by ethanol and drug intake.
European Journal of Clinical Investigation 19 (6), 549–555.
Williams, P.A., Cosme, J., Vinković, D.M., Ward, A., Angove, H.C., Day, P.J.,
Vonrhein, C., Tickle, I.J., Jhoti, H., 2004. Crystal structures of human
cytochrome P450 3A4 bound to metyrapone and progesterone. Science 305
(5684), 683–686.
Yang, C.S., Yoo, J.S., Ishizaki, H., Hong, J.Y., 1990. Cytochrome P450IIE1:
roles in nitrosamine metabolism and mechanisms of regulation. Drug
Metabolism Reviews 22 (2-3), 147–159.
Yoo, J.S., Guengerich, F.P., Yang, C.S., 1988. Metabolism of N-nitrosodialk-
ylamines by human liver microsomes. Cancer Research 48 (6), 1499–1504.
Peter, R., Böcker, R., Beaune, P.H., Iwasaki, M., Guengerich, F.P., Yang, C.S.,
1991. Hydroxylation of chlorzoxazone as a specific probe for human liver
cytochrome P-450IIE1. Chemical Research in Toxicology 3 (6), 566–573.
Phillips, A.H., Langdon, R.G., 1962. Hepatic triphosphopyridine nucleotide-
cytochrome c reductase: isolation, characterization, and kinetic studies.
Journal of Biological Chemistry, 237 (8), 2652–2660.