Marmoset pulmonary cytochrome P450 2F1 oxidizes biphenyl and 7-ethoxycoumarin and hepatic human P450 substrates

Article abstract of DOI:10.1080/00498254.2017.1354138

1. A potentially useful animal model for preclinical studies is the common marmoset (Callithrix jacchus). In this study, using reverse-transcription polymerase chain reaction from marmoset livers, we identified a novel cytochrome P450 (P450) 2F1 cDNA with an open reading frame of 1473 bp. 2. High sequence identities of 92-94% with primate P450 2 F amino acid sequences were indicated by deduced amino acid sequences of P450 2F1 cDNA. Phylogenetic analysis indicates that marmoset P450 2F1 is more congruent with primate P450 2 F forms than those of other species such as rodents. 3. Among five tissue types examined, abundant expression of marmoset P450 2F1 mRNA and P450 2F1 protein in lungs was shown. Cynomolgus monkey P450 2F1 mRNA was abundantly expressed in lungs as well as testes and ovaries in 10 tissue types. 4. Similar to those of humans and cynomolgus monkeys, marmoset P450 2F1 heterologously expressed in Escherichia coli membranes efficiently catalyzed 7-ethoxycoumarin O-deethylation and biphenyl hydroxylation, however unlike human P450 2F1, marmoset P450 2F1 exhibited hydroxylation activity toward coumarin and chlorzoxazone. 5. These findings indicated that P450 2F1 enzyme expressed in marmoset lungs and also catalyzed metabolism of xenobiotics, suggesting the importance of P450 2 F-dependent drug metabolism in marmoset lungs.

Full text of DOI:10.1080/00498254.2017.1354138

Xenobiotica
the fate of foreign compounds in biological systems
ISSN: 0049-8254 (Print) 1366-5928 (Online) Journal homepage: http://www.tandfonline.com/loi/ixen20
Marmoset pulmonary cytochrome P450 2F1
oxidizes biphenyl and 7-ethoxycoumarin and
hepatic human P450 substrates
Shotaro Uehara, Yasuhiro Uno, Toru Oshio, Takashi Inoue, Erika Sasaki &
Hiroshi Yamazaki
To cite this article: Shotaro Uehara, Yasuhiro Uno, Toru Oshio, Takashi Inoue, Erika
Sasaki & Hiroshi Yamazaki (2017): Marmoset pulmonary cytochrome P450 2F1 oxidizes
biphenyl and 7-ethoxycoumarin and hepatic human P450 substrates, Xenobiotica, DOI:
10.1080/00498254.2017.1354138
To link to this article: http://dx.doi.org/10.1080/00498254.2017.1354138
Accepted author version posted online: 08
Jul 2017.
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Date: 09 July 2017, At: 06:12

Marmoset pulmonary cytochrome P450 2F1 oxidizes biphenyl and 7-ethoxycoumarin
and hepatic human P450 substrates
1
2
1
3
Shotaro Uehara , Yasuhiro Uno , Toru Oshio , Takashi Inoue , Erika Sasaki 3,4, and
Hiroshi Yamazaki 1
1
Laboratory of Drug Metabolism and Pharmacokinetics, Showa Pharmaceutical University,
2
Machida, Tokyo 194-8543, Japan, Pharmacokinetics and Bioanalysis Center, Shin Nippon
3
Biomedical Laboratories, Ltd., Kainan, Wakayama, Japan,
Department of Applied
4
Developmental Biology, Central Institute for Experimental Animals, Kawasaki, Japan, and
Keio Advanced Research Center, Keio University, Minato-ku, Tokyo, Japan.
Running title: Cloning of marmoset cytochrome P450 2F1 cDNA
Shotaro Uehara and Yasuhiro Uno equally contributed.
Correspondence author: Hiroshi Yamazaki, PhD, Professor
Showa Pharmaceutical University, 3-3165 Higashi-tamagawa Gakuen, Machida, Tokyo
194-8543, Japan.
Phone: +81-42-721-1406; Fax: +81-42-721-1406. E-mail:
hyamazak@ac.shoyaku.ac.jp

Abstract
1. A potentially useful animal model for preclinical studies is the common marmoset
(Callithrix jacchus). In this study, using reverse-transcription polymerase chain reaction
from marmoset livers, we identified a novel cytochrome P450 (P450) 2F1 cDNA with an
open reading frame of 1473 bp.
2. High sequence identities of 92-94% with primate P450 2F amino acid sequences were
indicated by deduced amino acid sequences of P450 2F1 cDNA. Phylogenetic analysis
indicates that marmoset P450 2F1 is more congruent with primate P450 2F forms than
those of other species such as rodents.
3. Among five tissue types examined, abundant expression of marmoset P450 2F1 mRNA
and P450 2F1 protein in lungs was shown. Cynomolgus monkey P450 2F1 mRNA was
abundantly expressed in lungs as well as testes and ovaries in ten tissue types.
4. Similar to those of humans and cynomolgus monkeys, marmoset P450 2F1 heterologously
expressed in Escherichia coli membranes efficiently catalyzed 7-ethoxycoumarin
O-deethylation and biphenyl hydroxylation, however unlike human P450 2F1, marmoset
P450 2F1 exhibited hydroxylation activity toward coumarin and chlorzoxazone.
5. These findings indicated that P450 2F1 enzyme expressed in marmoset lungs and also
catalyzed metabolism of xenobiotics, suggesting the importance of P450 2F-dependent
drug metabolism in marmoset lungs.
Keywords: Common marmoset; CYP2F1; drug oxidation.

Introduction
Cytochrome P450s (P450s, EC 1.14.14.1) are the major enzymes that influence the
metabolism of many xenobiotic compounds, including environmental carcinogens and drugs.
Human P450 2F1 is one such catalyst, and involved in the bioactivation of pulmonary toxins,
including 3-methylindole, naphthalene, and styrene. The P450 2F subfamily contains a single
member in each species, which is primarily expressed in lungs; P450 2F1 in humans
(Nhamburo et al., 1990), P450 2F5 in gorillas (Chen et al., 2002), P450 2F3 in goats (Wang et
al., 1998), P450 2F4 in rats (Baldwin et al., 2005), and P450 2F2 in mice (Ritter et al., 1991).
Although the Old World primate cynomolgus monkey (Macaca fascicularis) has been
used for drug development and safety assessment, the common marmoset (Callithrix jacchus),
generally referred to as The New World primate, is an ideal animal model for preclinical
pharmacokinetics, toxicity, and assessment of metabolism of drugs because of their genetic
closeness and phenotypic similarities to humans, small body size, ease of handling, high
fertility, and early sexual maturity ('t Hart et al., 2012; Sasaki, 2015). Drug- and
xenobiotic-metabolizing enzymes have not been sufficiently analyzed in marmosets, nor
adequately compared with humans and cynomolgus monkeys.
Our group has identified more than twenty P450 forms belonging the P450 1-4 families
in marmosets. Their amino acid sequences and substrate selectivity have been found to be
similar to those of humans and cynomolgus monkeys (Uno et al., 2016). In this study, we
isolated novel P450 2F1 cDNA from marmoset lungs, analyzed sequence identity, gene
expression patterns, drug-metabolizing activity, and also compared its characteristics to the
corresponding human and cynomolgus monkey P450 2F forms.

Materials and Methods
Chemicals and tissue samples
Coumarin, 7-hydroxycoumarin, and biphenyl were purchased from Wako Pure Chemicals
(Osaka, Japan). 2- and 4-Hydroxybiphenyl were purchased from Tokyo Kasei (Tokyo, Japan).
7-Ethoxycoumarin and chlorzoxazone were purchased from Sigma-Aldrich (St. Louis, MO,
USA). 6-Hydroxychlorzoxazone was purchased from Toronto Research Chemicals (Toronto,
Canada). DNA oligonucleotides were synthesized by Sigma Chemical Co. (Tokyo, Japan).
Human P450 2F1 cDNA was purchased from NovoPro Bioscience (Shanghai, China). Pooled
liver microsomes from marmosets and humans were purchased from Corning Life Sciences
(Woburn, MA, USA). Pooled liver microsomes from cynomolgus monkeys and pooled lung
microsomes from humans were purchased from Xenotech (Lenexa, KS, USA). Lung
microsomes from marmosets and cynomolgus monkeys were prepared from lung tissues of
18 (10 males and 8 females, 2-6 years of age) and 6 (3 males and 3 females, 4-5 years of age)
animals, respectively. Marmoset tissues from brains, lungs, livers, kidneys, and jejunums
were collected from 20 marmosets (10 males and 10 females, 2-6 years of age) raised at the
Central Institution for Experimental Animals (Kawasaki, Japan). Cynomolgus monkey brains,
lungs, hearts, livers, kidneys, jejunums, adrenal glands, testes, ovaries, and uteri were
collected from 6 cynomolgus monkeys (3 males and 3 females from Indochina, 4-5 years of
age) at Shin Nippon Biomedical Laboratories, Ltd. (Kainan, Japan). The study was approved
by the Institutional Animal Care and Use Committee. Pooled marmoset tissue microsomes
were prepared as described previously (Uehara et al., 2015b). All other chemicals were of the
highest grade commercially available.

P450 2F1 cDNA cloning
Marmoset and cynomolgus monkey P450 2F1 cDNAs were isolated by reverse transcription
(RT)-polymerase chain reaction (PCR) using total RNA from lung tissues. Briefly, total
RNAs were extracted from lungs of marmosets and cynomolgus monkeys with RNeasy Mini
Kit (Qiagen, Valencia, CA). The first-strand cDNA was synthesized with 1.0 μg of total RNA
using the Superscript III First Strand Synthesis kit (Invitrogen, Carlsbad, CA, USA). PCR
was performed using the high-fidelity DNA polymerase KOD-Plus-Neo (Toyobo, Osaka,
Japan) with an ABI GeneAmp PCR System 9700 thermocycler (Applied Biosystems, Foster
City,
CA,
USA).
The
primers
used
and
were
cjCYP2F1(5rt2)
5-
5-
GATCCTACTCTCTCCTCAGCG-3
cjCYP2F1(3rt2)
CTCATCGCTCACAGGGGAATC-3 for marmoset P450 2F1, and mmCYP2F1 (5rt1)
5-GACGCCAGCAGCTGCCTTCACC-3 and mmCYP2F1 (3rt1)
5-GCCTCATTGCTCACAGGCGAATC-3 for cynomolgus monkey P450 2F1. Thermal
cycler conditions included an initial 2-minute denaturation at 94°C, followed by 30 cycles of
98°C for 15 seconds, 60°C for 30 seconds, and 68°C for 2 minutes, with a final 7-minute
extension at 68°C. For cynomolgus monkey P450 2F1, annealing was performed at 65°C for
30 seconds. The amplified fragments were cloned into pGEM-T easy vectors (Promega,
Madison, WI, USA) or pCR4 Blunt TOPO vectors (Invitrogen), and an inserted DNA
fragment was sequenced using ABI PRISM BigDye Terminator v3.0 Ready Reaction Cycle
Sequencing Kit (Applied Biosystems) with an ABI PRISM 3730 DNA Analyzer (Applied
Biosystems).
Bioinformatics
The homology of amino acid sequences was determined using BLAST (National Center for

Biotechnology Information). The multiple alignment of sequences was performed using a
Genetyx system (Software Development, Tokyo, Japan). The phylogenetic tree was created
using DNASIS Pro (Hitachi Software, Tokyo, Japan). The P450 amino acid sequences from
GenBank used for the analysis included; human P450 2F1 (NP_000765), gorilla P450 2F5
(AAL72278), rhesus monkey P450 2F1 (NP_001272197), goat P450 2F3 (NP_001274499),
rat P450 2F4 (NP_001274499), and mouse P450 2F2 (NP_031843). The marmoset P450 2F1
amino acid sequence was deduced from the cDNA identified in this study.
Quantitative real-time RT-PCR
The expression levels of P450 2F1 mRNA in marmoset and cynomolgus monkey tissues were
measured by real-time RT-PCR. Total RNA was extracted from marmoset and cynomolgus
monkey tissues using an RNeasy Mini Kit as described earlier. An equal amount of total RNA
was pooled from 3 male marmosets or 6 cynomolgus monkeys (3 males and 3 females) for
each tissue type, and pooled total RNA (1 µg) was reverse transcribed according to the
procedure described above. Real-time PCR was performed using Power SYBR Green PCR
Master Mix (Applied Biosystems, Foster City, CA) with an ABI PRISM 7300 sequence
detection system (Applied Biosystems). The PCR reaction mixture contained 400 nM of each
primer, 12.5 µl of Power SYBR Green PCR Master Mix, and 2 µl of RT products in a total
volume
of
25
µl.
The
PCR
primers
and
were
cjCYP2F1
(5qrt1)
(3qrt1)
5'-GGAGAGGAGTTTAGTGGCCG-3'
cjCYP2F1
5'-CTTCAGTTTTCCGCAGCTCC-3' for marmoset P450 2F1, mfCYP2F1 (5qrt1)
5'-ACCAACGCATCTTCCAGAACT-3' and mfCYP2F1 (3qrt1)
5'-CTCCTTCTCCTCTGCCATCT-3' for cynomolgus monkey P450 2F1, and mf&h_b-actin
(5qrt1) 5-GGAAATCGTGCGTGACATT-3 and mf&h_b-actin (3qrt1)
5-TTTCGTGGATGCCACAGG-3 for cynomolgus monkey β-actin. Levels of 18S ribosomal

RNA were measured using Eukaryotic 18S ribosomal RNA Endogenous Control (Applied
Biosystems) according to the manufacturer’s instructions. Thermal cycler conditions were
10 minutes at 95°C, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute.
All samples were analyzed in triplicate. The abundance of P450 2F1 mRNA in various tissues
was expressed as relative to 18S ribosomal RNA levels for marmosets and β-actin mRNA
levels for cynomolgus monkeys.
Immunoblotting
The expression of P450 2F1 protein in marmoset liver microsomes was analyzed by
immunoblotting with human P450 2F1 polyclonal antibodies [CYP2F1 (H-41), Santa Cruz
Biotechnology, Santa Cruz, CA]. In brief, recombinant P450 protein (0.1 pmol of P450/lane)
or tissue microsomes (40 μg/lane) were run on 10% sodium dodecyl sulphate-polyacrylamide
gel and the resolved proteins were transferred onto polyvinylidene difluoride membrane
(Merck Millipore, Billerica, MA). The membranes were blocked with 0.5% skim milk at
room temperature for 30 minutes, incubated with anti-human P450 2F1 antibodies (1:1000) at
room temperature for 1 hour, and then with goat anti-rabbit secondary antibodies (1:5000;
Santa Cruz Biotechnology) at room temperature for 20 minutes. P450 2F1 protein bands were
visualized by an ECL Prime Western Blotting Detection System (GE Healthcare,
Buckinghamshire, UK) according to the manufacturer’s instructions.
Heterologous expression of P450 2F protein in Escherichia coli
For enzyme assays, recombinant marmoset P450 2F1 was prepared by heterologous
expression in Escherichia coli using expression plasmids. The N-terminus modification was
carried
out
by
PCR
using
the
primers,
cjCYP2F1
(5exp3)
5'-CATATGGCTAAGAAAACGAGCTCTAAAGGTAAGCTGCCTCCAGGACCCAGA-3'

and cjCYP2F1(3exp1) 5'-TCTAGATTAGCGCGCGCGCAGGCACAGC-3' for marmoset
P450 2F1, cjCYP2F1 (5exp3) and mfCYP2F1(3exp1) 5'-
TCTAGATTAGTGCGGGCACAGGCACAGC-3' for cynomolgus monkey P450 2F1, and
cjCYP2F1 (5exp3) and hCYP2F1(3exp1)
5'-TCTAGATTAGCGCGGGCGCAGGCACAGC-3' for human P450 2F1. PCR product
was subcloned into the pCW vector containing human NADPH-P450 reductase cDNA via the
cloning sites NdeI and XbaI (underlined). The sequences of the inserts were verified by
DNA sequencing as described earlier. For cynomolgus monkey P450 2F1, the internal XbaI
site was mutated without changing the amino acid residue using QuikChange XL II kit
(Stratagene, La Jolla, CA) according to manufacturer’s instructions. Primers used were
mfCYP2F1_c780 (5qc1) 5'- ATTGTTTGTTTCTCACATGATTGACAAACAC-3' and
mfCYP2F1_c780 (3qc1) 5'-GTGTTTGTCAATCATGTGAGAAACAAACAAT-3'. The
heterologous expression in Escherichia coli, preparation of membrane fractions, and the
measurement of concentrations of P450 and NADPH-P450 reductase were performed as
described previously (Yamazaki et al., 2002).
Enzyme assays
As described previously (Uehara et al., 2015a), 7-ethoxycoumarin O-deethylation, coumarin
7-hydroxylation, and chlorzoxazone 6-hydroxylation activities by recombinant P450s and
liver microsomes from marmosets and humans were measured by high-performance liquid
chromatography. Biphenyl hydroxylation activities were measured as described previously
(Shimada et al., 2016) with some minor changes as follows. In brief, a typical incubation
mixture (0.25 mL) contained recombinant protein (10 pmol) or liver microsomes (0.125
mg/mL), an NADPH-generating system (0.25 mM NADP+, 2.5 mM glucose 6-phosphate,
0.25 units/mL glucose 6-phosphate dehydrogenase), and 100 μM biphenyl in 100 mM

potassium phosphate buffer (pH 7.4). The reaction was initiated by the addition of
NADPH-generating system. The reaction mixtures were incubated at 37°C for 20 min, and
terminated by adding 250 μL of methanol, and then 1.0 mL of a mixture of chloroform and
ethyl acetate (1:1, v/v). After centrifugation at 20,000g for 10 min, supernatants (50 μL) were
injected into a reversed-phase C18 column (Mightysil, 5 mm, 150×4.6 mm; Kanto Chemical,
Tokyo, Japan) with elution by a mobile phase (19% acetonitrile, 29% methanol, and 52%
12mM ammonium acetate buffer, pH 4.0) at 1.0 mL/min, monitoring at UV 254 nm and
fluorescence with an excitation wavelength of 242 nm and emission wavelength of 380 nm.
Results and Discussion
The potential to use marmosets in pharmacokinetic and pharmacological studies, although
high, is diminished by limited information being available regarding their drug-metabolizing
enzymes. We have identified and characterized more than 20 hepatic P450 in marmosets, and
indicated that the P450s have similar tissue expression patterns and enzymatic properties to
those of humans (Shimizu et al., 2014; Uno et al., 2016). However, extrahepatic P450s still
need to be fully analyzed in marmosets. Analysis of the marmoset genome sequences using
BLAT (UCSC Genome Bioinformatics, University of California, Santa Cruz, CA) revealed
the novel marmoset P450 2F (P450 2F1) gene which is highly homologous to human P450
2F1 cDNA. Implementing RT-PCR with the gene-specific primers enabled us to isolate P450
2F1 cDNA containing the entire coding region (1668 bp) from marmoset liver. Deduced
amino acids (491 residues) contained the primary sequence structure characteristic of P450
proteins, such as substrate recognition sites (SRSs) and the heme-binding region (Fig. 1).
When comparing P450 2F cDNA sequences, marmoset P450 2F1 showed high amino acid
sequence identity to primate P450 2F forms at 92-94%, whereas those of rodent P450 2Fs

were significantly less similar at 81-82% (Table 1). Phylogenetic analysis of P450 2F amino
acid sequences conferred that marmoset and human, together with cynomolgus monkey and
rhesus monkey P450 2F1 forms were closely clustered, unlike goat, rat, and mouse P450 2F
forms (Fig. 2).
To determine the expression pattern of P450 2F1 mRNA in marmoset tissues, real-time
RT-PCR was performed with the gene-specific primers in brain, kidney, liver, lung, and small
intestine. Marmoset P450 2F1 mRNA level was notably abundant in lungs (Fig. 3A), also
clearly apparent by the single band (approximately 50 kDa) detected in lung microsomes with
anti-human P450 2F1 antibodies which cross-reacted recombinant marmoset P450 2F1
proteins (Fig. 4). Species differences of tissue distribution of P450 2F forms are considered in
various experimental animals. Marmoset P450 2F1 mRNAs were expressed at a high level in
lungs compared to other tissues (Fig 3A). Human P450 2F1 mRNA was mainly expressed in
the respiratory tract (lung and trachea), followed by testis, and very low or none in the liver.
(Nishimura et al., 2003; Bieche et al., 2007). Interestingly cynomolgus monkey P450 2F1
mRNA, although abundantly expressed in lungs was significantly more so in testes and
ovaries (reproductive organs), unlike those of humans where it was 100-fold higher in lungs
than testis (Fig 3B) (Nishimura et al., 2003). In mice and rats, P450 2F forms were
abundantly expressed in livers, lungs, and nasal olfactory mucosa (Baldwin et al., 2004;
Renaud et al., 2011). These results indicated that the abundant expression of P450 2F1 was
conserved among marmosets, cynomolgus monkeys and humans.
Using some typical human P450 substrates, including 7-ethoxycoumarin, coumarin,
biphenyl, and chlorzoxazone, enzyme assays were carried out to investigate the
drug-metabolizing activity of marmoset P450 2F1. Marmoset lung microsomes catalyzed
7-ethoxycoumarin O-deethylation and biphenyl hydroxylation at low rates, compared to

marmoset liver microsomes. Recombinant marmoset P450 2F1 catalyzed 7-ethoxycoumarin
O-deethylation, coumarin 7-hydroxylation, biphenyl hydroxylation, and chlorzoxazone
6-hydroxylation (Table 2), indicating that 7-ethoxycoumarin O-deethylation and biphenyl
hydroxylation activities by P450 2F1 enzymes were conserved among marmosets,
cynomolgus monkeys, and humans. The kinetics analysis indicated that the Km and Vmax for
7-ethoxycoumarin O-deethylation by P450 2F enzymes were largely different among
marmosets, cynomolgus monkeys, and humans (Table 3 and Fig. 5) which further points to
differences existing among species in the substrate selectivity of P450 2F enzymes. Goat
P450 2F3, as another example of substrate selectivity, metabolizes human P450 2F1
substrates, naphthalene and 3-methylindole, but not 7-ethoxycoumarin, unlike human P450
2F1 (Wang et al., 1998). Studies comparing of the dehydrogenation of 3-methylindole by
P450 2F orthologs from humans (P450 2F1), mice (P450 2F2), and goats (P450 2F3)
demonstrated relatively large differences in kinetic parameters (Lanza et al., 1999). To
understand the congruencies and inconsistencies of the P450 2F-dependent metabolism in
marmosets, humans, and other experimental animals, further analysis of enzymatic
characteristics and substrate specificity is needed. Previous reports indicated that human P450
2F1, beyond metabolizing classical P450 substrates such as 7-ethoxycoumarin,
propoxycoumarin, and pentoxyresorufin, also metabolize pulmonary toxicants or
procarcinogens including naphthalene, styrene, 3-methylindole, and benzene (Nhamburo et
al., 1990; Nakajima et al., 1994; Lanza et al., 1999; Sheets et al., 2004). Marmoset P450 2F1
also metabolized the air pollutant biphenyl, suggesting that their lung P450 2F1 may be
similar to humans in the detoxification of environmental toxicants. There is a very likely
significant benefit to investigating the metabolism of various pulmonary toxicants and
procarcinogens by marmoset P450 2F1 in the future.
In summary, we have identified in marmosets a novel P450 2F1 highly homologous to

that of humans. This study of P450 2F1 showed: amino acid sequence identities of 93% with
similar tissue expression patterns to human, catalysis of 7-ethoxycoumarin O-deethylation
and biphenyl hydroxylation, also congruently catalyzed by human P450 2F1, and suggested
that the metabolism of drugs and environmental chemicals in marmoset lungs are similar
among human and cynomolgus monkey P450 2F1, thus making marmosets a contending
animal model for preclinical studies.
Acknowledgments
The authors thank Drs. Elizabeth Gillam, Makiko Shimizu, and Norie Murayama for their
technical help and Mr. Lance Bell for his advice on English writing.
Declaration of interest
This work resulted from the "Construction of System for Spread of Primate Model Animals"
initiative under the Strategic Research Program for Brain Sciences of the Japan Agency for
Medical Research and Development. Shotaro Uehara was also supported partly by the Japan
Society for the Promotion of Science Grant-in-Aid for Young Scientists B [17K15520]. The authors
alone are responsible for the content and for writing the article and report no declarations of
interest.

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Yamazaki H, Nakamura M, Komatsu T, Ohyama K, Hatanaka N, Asahi S, Shimada N,
Guengerich FP, Shimada T, Nakajima M, and Yokoi T (2002) Roles of NADPH-P450
reductase and apo- and holo-cytochrome b5 on xenobiotic oxidations catalyzed by 12
recombinant human cytochrome P450s expressed in membranes of Escherichia coli.
Protein Expr Purif 24:329-337.

Table 1
Sequence identity of P450 2F1 cDNA and amino acids as compared to P450 2Fs of other
species.
Identity (%)
Species
P450
cDNA
94
N.A.
94
94
85
Amino acids
Human
Gorilla
Cynomolgus monkey
Rhesus monkey
Goat
2F1
2F5
2F1
2F1
2F3
2F4
2F2
93
93
94
92
83
82
81
Rat
Mouse
83
83
N.A., not applicable

Table 2
Xenobiotic-metabolizing activities by tissue microsomes from marmosets, cynomolgus
monkeys, and humans, and recombinant P450 2F1 enzymes.
7-Ethoxycoumarin
O-deethylation
pmol/min/mg protein
122 ± 4
Coumarin
7-hydroxylation
pmol/min/mg protein
550 ± 11
Biphenyl
2- and 4-hydroxylation
pmol/min/mg protein
210 ± 14
Chlorzoxazone
Enzyme source
6-hydroxylation
Microsomes
pmol/min/mg protein
Human liver
142 ± 41
Cynomolgus monkey liver
Marmoset liver
2800 ± 24
1400 ± 19
295 ± 23
352 ± 68
144 ± 8
<1.3
210 ± 17
157 ± 51
Human lung
3.8 ± 0.4
<0.32
2.6 ± 0.4
<12
Cynomolgus monkey lung
Marmoset lung
1.8 ± 0.1
0.38 ± 0.02
<0.32
2.2 ± 0.7
<12
3.7 ± 1.6
3.5 ± 0.4
<12
Recombinant P450
Human P450 2F1
Cynomolgus monkey
P450 2F1
pmol/min/nmol P450
49 ± 1
pmol/min/nmol P450
<1.3
pmol/min/nmol P450
65 ± 28
pmol/min/nmol P450
<72
<72
1.6 ± 0.2
2.9 ± 0.6
29 ± 7
Marmoset P450 2F1
57 ± 1
8.1 ± 0.4
74 ± 20
752 ± 41
Each catalytic activity was measured at a substrate concentration of 100 μM
7-ethoxycoumarin, 100 μM coumarin, 100 μM biphenyl, and 100 μM chlorzoxazone. Data
represent the mean ± standard deviation of triplicate determinations.

Table 3
Kinetic analyses for 7-ethoxycoumarin O-deethylation by liver microsomes from
marmosets and cynomolgus monkeys, and recombinant P450 2F1 enzymes.
Enzyme source
Km
μM
Vmax
pmol/min/mg protein
64 ± 11
Vmax/Km
Microsomes
μL/min/mg protein
Human lung
950 ± 280
760 ± 250
460 ± 140
μM
0.067
Cynomolgus monkey lung
Marmoset lung
11 ± 2
0.015
5.4 ± 0.8
0.012
Recombinant P450
Human P450 2F1
Cynomolgus monkey P450 2F1
Marmoset P450 2F1
pmol/min/nmol P450
180 a
μL/min/nmol P450
NA
NA
NA
NA
30 a
209 ± 72
145 ± 18
0.69
Kinetic parameters were determined by non-linear regression analysis (mean ± standard error,
n = 14 points of substrate concentrations of 1.7-1250 μM, duplicate determinations)
employing the equation, v = Vmax × [S]/(Km + [S]) for Michaelis-Menten equation using
Kaleidagraph software (Synergy Software, Reading, PA). a Activities at 1250 μM, NA, Not
available.

Legends for figures
Fig. 1. Comparison of P450 2F1 amino acid sequences from primates. P450 2F amino
acid sequences from marmosets (cj), humans (h), gorillas (g), cynomolgus monkeys (mf), and
rhesus monkey (mm) were aligned using Genetyx. Six substrate recognition sites (SRS) and a
heme-binding site are shown by solid and broken lines, respectively. Amino acid residues
conserved and roughly conserved among the five species are shown by asterisks and dots,
respectively, under amino acid alignment.
Fig. 2. A phylogenetic tree of P450 2F amino acid sequences from mammalians. A
phylogenetic tree was created using the neighbor-joining method using P450 2F amino acid
sequences of marmoset (cj), human (h), gorilla (g), cynomolgus monkey (mf), rhesus monkey
(mm), goat (c), rat (r), and mouse (m). Human P450 2A13 was used as an outgroup. The
scale bar represents a phylogenetic distance of 0.1 amino acid substitutions per site.
Fig. 3. Tissue distribution of P450 2F1 mRNA in marmosets and cynomolgus monkeys.
Expression levels of marmoset P450 2F1 mRNA in each pooled tissue from marmosets and
cynomolgus monkeys were measured by quantitative reverse-transcription real-time PCR,
and normalized with 18S rRNA or β-actin mRNA levels, respectively. P450 2F1 mRNA
levels in marmoset and cynomolgus monkey lungs were adjusted to 1, and the relative P450
2F1 mRNA expression levels are shown for other tissues. Each data was mean  standard
deviations of triplicate determinations.

Fig. 4. Detection of marmoset P450 2F1 proteins by immunoblotting. Recombinant
P450 2F1 protein (0.1 pmol of P450/lane) was detected by immunoblotting using anti-human
P450 2F1 antibodies cross-reacted with recombinant marmoset P450 2F1 protein.
Immunoreactive bands of approximately 50 kDa were detected in marmoset lung microsomes
(40 μg/lane). Protein disulfide isomerase (PDI) is used in membrane fraction loading control.
Fig. 5. Kinetic parameters for 7-ethoxycoumarin O-deethylation by lung microsomes
from marmosets and cynomolgus monkeys, and recombinant P450 2F1 enzymes. Kinetic
analyses were performed for 7-ethoxycoumarin O-deethylation by lung microsomes (A) and
recombinant P450 2F1 enzymes (B) . Circles, marmosets; triangles, cynomolgus monkeys;
squares, humans.

Fig. 1
cjCYP2F1
mfCYP2F1
mmCYP2F1
gCYP2F5
hCYP2F1
1:MDSISTAMLL LLLALICLLL TLSSRDKGKL PPGPRPLPLL GNLLLLRSQD MLTSLTELSK EYGSMYTVYL GSRRVVVLSG FQAVKEALVD QGEEFSGRGD 100
1:MDSISTAILL LLLALVCLLL TLSSRDKRKL PPGPRPLPLL GNLLLLRSQN MLTSLTQLSK EYGSVYTVHL GPRRVVVLSG YQAVKEALVD QGEEFSGRGD 100
1:MDSISTAILL LLLALVCLLL TLSSRDKXKL PPGPRPLPLL GNLLLLRSQN MLTSLTQLSK EYGSVYTVHL GPRRVVVLSG YQAVKEALVD QGEEFSGRGD 100
1:MDSVSTAILL LLLALICLLL TLSSRDKGKL PPGPRPLPLL GNLLLLRSQD MLTSLTKLSK EYGSMYTVHL GPRRVVVLSG YQAVKEALVD QGEEFSGRRD 100
1:MDSISTAILL LLLALVCLLL TLSSRDKGKL PPGPRPLSIL GNLLLLCSQD MLTSLTKLSK EYGSMYTVHL GPRRVVVLSG YQAVKEALVD QGEEFSGRGD 100
***.***.** *****.**** *******.** *******..* ******.**. ****** *** ****.***.* *.******** .********* ********.*
SRS-1
cjCYP2F1 101:YPVFFNFTKG NGIAFSNGDR WKILRRFSIQ ILRGFGMGKA SIEERILEEG SFLLAELRKT EGEPFDPTFV LSRSVSNIIC SVLFGSRFDY EDERLLTIIR 200
mfCYP2F1 101:YPVFFNFTKG NGIAFSNGDR WKVLRRFSIQ ILWNFGMGKR SIEERILEEG SFLLAELRKT EGEPFDPTFV LSRSVSNIIC SVLFGSRFDY DDERLLTVIR 200
mmCYP2F1 101:YPVFFNFTKG NGIAFSNGDR WKVLRRFSIQ ILRNFGMGKR SIEERILEEG SFLLAELRKT EGEPFDPTFV LSRSVSNIIC SVLFGSRFDY DDERLLTVIR 200
gCYP2F5 101:FPAFFNFTKG NGIAFSNGDR WKVLRRFSIQ ILRNFGMGKR SIEERILEEG SFLLAELRKT EGEPFDPTFV LSRSVSNIIC SVLFGSRFDY DDERLLTIIR 200
hCYP2F1 101:YPAFFNFTKG NGIAFSSGDR WKVLRQFSIQ ILRNFGMGKR SIEERILEEG SFLLAELRKT EGEPFDPTFV LSRSVSNIIC SVLFGSRFDY DDERLLTIIR 200
.*.******* ******.*** **.**.**** **..*****. ********** ********** ********** ********** ********** .******.**
SRS-2
SRS-3
cjCYP2F1 201:LINDNFQLMS SPWGELYNIF PSLLDWVPGP HRRIFQNFKC LRDLIVHSVH DHQASLDPRS PRDFIDCFLT KMAEEKEDPQ SHFHMDTLLM TTHNLLFGGT 300
mfCYP2F1 201:LINDNFQIMS SPWGELYNIF PSLLDWVPGP HQRIFQNFKR LRDLIAHSVH DHQASLDPRS PRDFIDCFLT KMAEEKEDPL SHFHMDTLLM TTHNLLFGGT 300
mmCYP2F1 201:LINDNFQIMS SPWGELYNIF PSLLDWVPGP HQRIFQNFKR LRDLIAHXVH DQQASLDPRS PRDFIDCFLT KMAEEKEDPL SHFHMDTLLM TTHNLLFGGT 300
gCYP2F5 201:HINDNFQIMS SPWGELYDIF PSLLDWVPGP HQRIFQNFKC LRDLIAHSVH DHQASLDPRS PRDFIDCFLT KMAEENEDPL SHFHMDTLLM TTHNLLFGGT 300
hCYP2F1 201:LINDNFQIMS SPWGELYDIF PSLLDWVPGP HQRIFQNFKC LRDLIAHSVH DHQASLDPRS PRDFIQCFLT KMAEEKEDPL SHFHMDTLLM TTHNLLFGGT 300
.******.** *******.** ********** *.*******. *****.*.** *.******** *****.**** *****.***. ********** **********
SRS-4
SRS-5
cjCYP2F1 301:ETVSTTLRHA FLALMKYPKV QARVQEEIDL VVGRARLPAL QDRAAMPYTD AVIHEVQRFA NVIPMNLPHR VTRDTVFRGF LIPKGTDIIT LLNTVHHDPS 400
mfCYP2F1 301:ETVGTTLRHA FLALMKYPKV QARVQEEIDL VVGRTRLPTL EDRAAMPYTD AVIHEVQRFA DIIPMNLPHR VIRDTAFRGF LIPKGTDIIT LLNTVHYDPS 400
mmCYP2F1 301:ETVGTTLRHA FLALMKYPKV QARVQEEIDL VVGRTRLPTL EDRAAMPYTD AVIHEVQRFA DIIPMNLPHR VIRDTAFRXF LIPKGTDIIT LLNTVHYDPS 400
gCYP2F5 301:ETVGTTLRYA FLALMKYPKV QARVQEEIDL VVGRARLPAL KDRAAMPYTD AVIHEVQRFA DIIPMSLPHR VTRDTAFRGF LIPKGTDIIT LLNTVHYDPS 400
hCYP2F1 301:KTVSTTLHHA FLALMKYPKV QARVQEEIDL VVGRARLPAL KDRAAMPYTD AVIHEVQRFA DIIPMNLPHR VTRDTAFRGF LIPKGTDVIT LLNTVHYDPS 400
.**.***..* ********** ********** ****.***.* ********* ********** ..***.**** *.***.**.* *******.** ******.***
SRS-6
cjCYP2F1 401:QFLTPQEFNP EHFLDASQSF KKSPAFMPFS AGRRLCLGES LGRMELFLYL TAILQSFSLQ PLGAPEDIDL TPLSSGLGNL PRPFQLCLRA R
mfCYP2F1 401:QFLTPQEFNP EHFLDANQSF KKSPAFMPFS AGRRLCLGES LARMELFLYL TAILQSFSLQ PLGAPEDIDL TPLSSGLGNL PRPFQLCLCP H
mmCYP2F1 401:QFLXPQEFNP EHFLDANQSF KKSPAFMPFS AGRRLCLGES LARMELFLYL TAILQSFSLQ PLGAPEDIXL TPLSSGLGNL PRXFQLCLCP R
gCYP2F5 401:QFLTPQEFNP EHFLDANQSF KKSPAFMPFS AGRRLCLGES LARMELFLYL TAILQSFSLQ PLGAPKDIDL TPLSSGLGNL PRPFQLCLRP R
hCYP2F1 401:QFLTPQEFNP EHFLDANQSF KKSPAFMPFS AGRRLCLGES LARMELFLYL TAILQSFSLQ PLGAPEDIDL TPLSSGLGNL PRPFQLCLRP R
***.****** ******.*** ********** ********** *.******** ********** *****.**.* ********** **.*****.. .
491
491
491
491
491

Fig. 2
gCYP2F5
hCYP2F1
mfCYP2F1
mmCYP2F1
cjCYP2F1
cCYP2F3
mCYP2F2
rCYP2F4
hCYP2A13
0.1

Fig. 3
A
1.0
0.5
0
Marmoset tissues
B
1.0
0.5
0
Cynomolgus monkey tissues

Fig. 4
Marmoset tissue microsomes
50 kDa
P450 2F1
PDI

Fig. 5
40
30
20
10
0
200
150
100
50
A
B
0
0
250
500
750 1000 1250
0
250
500
750 1000 1250
[7-Ethoxycoumarin], μM
[7-Ethoxycoumarin], μM