Structure-Function Studies of Naphthalene, Phenanthrene, Biphenyl, and Their Derivatives in Interaction with and Oxidation by Cytochromes P450 2A13 and 2A6

Article abstract of DOI:10.1021/acs.chemrestox.6b00083

Naphthalene, phenanthrene, biphenyl, and their derivatives having different ethynyl, propynyl, butynyl, and propargyl ether substitutions were examined for their interaction with and oxidation by cytochromes P450 (P450) 2A13 and 2A6. Spectral interaction studies suggested that most of these chemicals interacted with P450 2A13 to induce Type I binding spectra more readily than with P450 2A6. Among the various substituted derivatives examined, 2-ethynylnaphthalene, 2-naphthalene propargyl ether, 3-ethynylphenanthrene, and 4-biphenyl propargyl ether had larger ?A_max/K_s values in inducing Type I binding spectra with P450 2A13 than their parent compounds. P450 2A13 was found to oxidize naphthalene, phenanthrene, and biphenyl to 1-naphthol, 9-hydroxyphenanthrene, and 2-And/or 4-hydroxybiphenyl, respectively, at much higher rates than P450 2A6. Other human P450 enzymes including P450s 1A1, 1A2, 1B1, 2C9, and 3A4 had lower rates of oxidation of naphthalene, phenanthrene, and biphenyl than P450s 2A13 and 2A6. Those alkynylated derivatives that strongly induced Type I binding spectra with P450s 2A13 and 2A6 were extensively oxidized by these enzymes upon analysis with HPLC. Molecular docking studies supported the hypothesis that ligand-interaction energies (U values) obtained with reported crystal structures of P450 2A13 and 2A6 bound to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, indole, pilocarpine, nicotine, and coumarin are of use in understanding the basis of possible molecular interactions of these xenobiotic chemicals with the active sites of P450 2A13 and 2A6 enzymes. In fact, the ligand-interaction energies with P450 2A13 4EJG bound to these chemicals were found to relate to their induction of Type I binding spectra.

Full text of DOI:10.1021/acs.chemrestox.6b00083

Article
pubs.acs.org/crt
Structure−Function Studies of Naphthalene, Phenanthrene,
Biphenyl, and Their Derivatives in Interaction with and Oxidation by
Cytochromes P450 2A13 and 2A6
,
†
,†
‡
§
∥
Tsutomu Shimada,* Shigeo Takenaka,* Kensaku Kakimoto, Norie Murayama, Young-Ran Lim,
∥
⊥
,§
,#
Donghak Kim, Maryam K. Foroozesh, Hiroshi Yamazaki,* F. Peter Guengerich,*
and Masayuki Komori*
,†
†Laboratory of Cellular and Molecular Biology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University,
-58 Rinku-Orai-Kita, Izumisano, Osaka 598-8531, Japan
1
‡
§
∥
Osaka Prefectural Institute of Public Health, 1-3-69 Nakamichi, Higashinari-ku, Osaka 537-0025, Japan
Laboratory of Drug Metabolism and Pharmacokinetics, Showa Pharmaceutical University, Machida, Tokyo 194-8543, Japan
Department of Biological Sciences, Konkuk University, Seoul 143-701, Republic of Korea
⊥
Department of Chemistry, Xavier University of Louisiana, New Orleans, Louisiana 70125, United States
#
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, United States
S Supporting Information
*
ABSTRACT: Naphthalene, phenanthrene, biphenyl, and their derivatives having different
ethynyl, propynyl, butynyl, and propargyl ether substitutions were examined for their interaction
with and oxidation by cytochromes P450 (P450) 2A13 and 2A6. Spectral interaction studies
suggested that most of these chemicals interacted with P450 2A13 to induce Type I binding
spectra more readily than with P450 2A6. Among the various substituted derivatives examined,
2
-ethynylnaphthalene, 2-naphthalene propargyl ether, 3-ethynylphenanthrene, and 4-biphenyl
propargyl ether had larger ΔA /K values in inducing Type I binding spectra with P450 2A13
max
s
than their parent compounds. P450 2A13 was found to oxidize naphthalene, phenanthrene, and
biphenyl to 1-naphthol, 9-hydroxyphenanthrene, and 2- and/or 4-hydroxybiphenyl, respectively,
at much higher rates than P450 2A6. Other human P450 enzymes including P450s 1A1, 1A2,
1
B1, 2C9, and 3A4 had lower rates of oxidation of naphthalene, phenanthrene, and biphenyl
than P450s 2A13 and 2A6. Those alkynylated derivatives that strongly induced Type I binding
spectra with P450s 2A13 and 2A6 were extensively oxidized by these enzymes upon analysis
with HPLC. Molecular docking studies supported the hypothesis that ligand-interaction
energies (U values) obtained with reported crystal structures of P450 2A13 and 2A6 bound to 4-(methylnitrosamino)-1-(3-
pyridyl)-1-butanone, indole, pilocarpine, nicotine, and coumarin are of use in understanding the basis of possible molecular
interactions of these xenobiotic chemicals with the active sites of P450 2A13 and 2A6 enzymes. In fact, the ligand-interaction
energies with P450 2A13 4EJG bound to these chemicals were found to relate to their induction of Type I binding spectra.
1
2,13
INTRODUCTION
enzymes has been reported in humans
and also in Rhesus
■
14
monkeys. Metabolism of phenanthrene and biphenyl has also
been reported in various laboratory animals;
detailed studies on the roles of human P450s 2A13 and 2A6 in
the metabolism of naphthalene, phenanthrene, and biphenyl
have not been reported, except that Fukami et al. showed that
P450 2A13 can catalyze the oxidation of naphthalene in
Naphthalene, phenanthrene, and biphenyl are aromatic hydro-
carbon contaminants found in the environment, and the former
two chemicals belong to the class of polycyclic aromatic
hydrocarbons (PAHs) that includes toxic and carcinogenic
compounds such as benzo[a]pyrene, 7,12-dimethylbenz[a]-
15−22
however,
1
−5
anthracene, and benzo[c]phenanthrene.
These carcinogenic
13
humans.
PAHs have been shown to require metabolic activation by so-
called xenobiotic-metabolizing enzymes, such as cytochromes
P450 (P450), to evoke their toxic and carcinogenic responses in
laboratory animals as well as in humans.
Naphthalene has been shown to cause toxic responses in
several animal species and is known to require bioactivation by
P450 2F2 in mice to elicit toxicity.
Our previous studies have shown that naphthalene,
phenanthrene, biphenyl, and their substituted derivatives
induce Type I binding spectra with P450s 2A13 and 2A6,
suggesting that these chemicals may be oxidized by these P450
6
−8
23
enzymes. In this study, we studied how these chemicals are
9
,10
11
Hu et al. also reported
that P450 2A5 plays an important role in olfactory mucosal
Received: March 13, 2016
toxicity in mice. Metabolism of naphthalene by different P450
©
XXXX American Chemical Society
A
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Figure 1. Structures of chemicals used in this study.
oxidized by P450s 2A13 and 2A6 and whether there are
structure−function relationships for interacting with and
biotransformation by these enzymes, based on analysis with
HPLC. We used naphthalene and six substituted derivatives,
phenanthrene and six substituted derivatives, and biphenyl and
nine substituted derivatives for spectral interaction with and
metabolism by P450s 2A13 and 2A6. Other P450 enzymes
human 1A1, 1A2, 1B1, 2C9, and 3A4were also used to study
the oxidation of parent compounds naphthalene, phenanthrene,
and biphenyl. Docking simulations of the interactions of P450s
Enzymes. Human P450s, NADPH-P450 reductase, and cyto-
chrome b enzymes used in this study were expressed in Escherichia coli
5
2
3−25
as described previously.
Bacterial bicistronic P450s 1A2, 1B1
(
1B1.3, L432 V), 2A6, 2A13, 2C9, and 3A4 coexpressing human
NADPH-P450 reductase were also prepared, and E. coli membranes
were suspended in 10 mM Tris-HCl buffer (pH 7.4) containing 1.0
mM EDTA and 20% glycerol (v/v). Human P450s 1A1, 2A6, and
2
A13 and NADPH-P450 reductase were purified from membranes of
2
3−25
recombinant E. coli as described elsewhere.
Catalytic activities
(nmol of product formed/min/nmol P450) for these eight P450
enzymes were determined using typical model substrates and were
−1
found to be 35, 3.2, and 14 min for 7-ethoxyresorufin O-deethylation
2
A13 and 2A6 and these chemicals were also examined to
−
1
by P450s 1A1, 1A2, and 1B1, respectively; 3.3 and 1.4 min for
coumarin 7-hydroxylation by P450s 2A6 and 2A13, respectively; 3.5
visualize how these chemicals interact with these P450
enzymes.
−1
min for flurbiprofen 4′-hydroxylation by P450 2C9; and 3.2 and 6.5
−1
min for midazolam 1′- and 4′-hydroxylation, respectively, by P450
3A4.
EXPERIMENTAL PROCEDURES
Chemicals. Naphthalene, phenanthrene, and biphenyl were
obtained from Sigma-Aldrich (St. Louis, MO) or Wako Pure Chemical
■
Spectral Binding Titrations. Purified P450s 2A13 and 2A6 were
diluted to 1.0 μM in 0.10 M potassium phosphate buffer (pH 7.4)
containing 20% glycerol (v/v), and binding spectra were recorded with
subsequent additions of chemicals in a JASCO V-550 or OLIS-Aminco
DW2a spectrophotometer (Online Instrument Systems, Bogart, GA)
(Osaka, Japan) (Figure 1). 1-Naphthol, 4-naphthol, 9-hydroxyphenan-
threne, and 2- and 4-hydroxybiphenyl were also obtained from Wako
Pure Chemical. Acetylenic PAHs and biphenyl derivatives, including 2-
ethynylnaphthalene (2EN), 1-naphthalene methyl propargyl ether
2
3−25
as described previously.
Briefly, the chemicals were added to the
(
1NMPE), 1-naphthalene ethyl propargyl ether (1NEPE), 2-
buffer with or without the P450, and the spectra were recorded
between 350 and 500 (or 700) nm. The substrate binding spectra were
obtained by subtracting the blank spectra (in the absence of the P450)
from the P450 spectra (in the presence of the P450). Spectral
naphthalene propargyl ether (2NPE), 2-naphthalene methyl propargyl
ether (2NMPE), 2-naphthalene ethyl propargyl ether (2NEPE), 2-
ethynylphenanthrene (2EPh), 3-ethynylphenanthrene (3EPh), 9-
ethynylphenanthrene (9EPh), 2-(1-propynyl)phenanthrene (2PPh),
dissociation constants (K ) were estimated using GraphPad Prism
s
3
-(1-propynyl)phenanthrene (3PPh), 9-(1-propynyl)phenanthrene
software (GraphPad Software, San Diego, CA), using either hyperbolic
(
9PPh), 4-ethynylbiphenyl (4EB), 4-propynylbiphenyl (4PB), 4-
plots or quadratic fits in cases of tight binding.
butynylbiphenyl (4BuB), 2-biphenyl propargyl ether (2BPE), 4-
biphenyl propargyl ether (4BPE), 2-biphenyl methyl propargyl ether
Oxidation of Naphthalene. Oxidative metabolism of naphthalene
by P450s 1A2, 1B1, 2A6, 2A13, 2C9, and 3A4 was determined in a
standard incubation mixture (0.25 mL) containing 25 pmol of P450
(in bicistronic membranes), 50 μM naphthalene, and an NADPH-
generating system according to the method described by Fukami et
(
2BMPE), 4-biphenyl methyl propargyl ether (4BMPE), 2,2′-biphenyl
dipropargyl ether (22BDPE), and 4,4′-biphenyl dipropargyl ether
2
3−30
(
44BDPE) (Figure 1), were synthesized as described previously.
1
3
These substituted chemicals have been used to study the mechanisms
al. (The chemicals were dissolved in (CH ) SO as 10 mM stock
3 2
of inhibition of human P450 enzymes, including P450s 2A13 and
solutions and diluted, with the organic solvent concentration ≤ 0.5%,
v/v.). In reconstitution experiments, P450 membranes were replaced
by purified P450 1A1 (25 pmol), NADPH-P450 reductase (50 pmol),
23−30
2
A6.
Other chemicals and reagents used in this study were obtained from
the sources described previously and were of the highest quality
and L-α-dilauroyl-syn-glycero-3-phosphocholine (50 μg) as described
23−25
23−25
commercially available.
previously.
Incubation was carried out at 37 °C for 20 min,
B
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Figure 2. Spectral changes produced by the interaction of naphthalene (A), phenanthrene (B), and biphenyl (C) with P450 2A13. The lower
portion of each figure indicates the difference spectra obtained.
Figure 3. Comparison of spectral changes (spectral binding efficiency, ΔA /K ratio) from the interaction of various chemicals with P450s 2A6 and
max
s
2A13.
following a preincubation time of 1 min. Reactions were terminated by
C18-5 octadecylsilane column (2.0 mm × 150 mm) (Wako Pure
Chemical) with UV detection at 254 nm and fluorescence detection
with an excitation wavelength of 242 nm and emission wavelength of
380 nm. Elution of chemicals and their metabolites utilized a linear
gradient from 20 to 100% CH OH (v/v, in H O) for 25 min, followed
adding 100 μL of CH CN, and following centrifugation, the upper
3
layer was subjected to HPLC using a Mightsil RP-18 C18 GP column.
(
Note that naphthalene and its metabolites are volatile under a
nitrogen stream after these chemicals are extracted with organic
solvents.) The mobile phase used was 30% CH CN (v/v, in H O)
3
2
by holding at 100% CH OH for 5 min, with a flow rate of 0.2 mL/min.
3
2
3
containing 0.01% H PO (w/v).
Other Enzyme Assays. Coumarin 7-hydroxylation, 7-ethoxyr-
esorufin O-deethylation, flurbiprofen 4-hydroxylation, and midazolam
1′- and 4′-hydroxylation activities were determined using bicistronic
bacterial membrane or reconstituted monooxygenase systems as
3
4
Oxidation of Phenanthrene and Biphenyl and Their
Derivatives and Naphthalene Derivatives. Oxidative metabolism
of phenanthrene and biphenyl and their derivatives (and also
naphthalene derivatives) was determined in a standard reaction
mixture (final volume of 0.25 mL) as described above. After incubation
at 37 °C for 20 min, extraction was done by adding 0.25 mL of cold
CH OH and then 0.5 mL of a mixture of CHCl and ethyl acetate
2
3−25,31−33
described previously.
Docking Simulations into Human P450 Enzymes. Crystal
34
structures of P450 2A13 bound to NNK (PDB 4EJH), indole (PDB
3
5
36
34
2P85), pilocarpine (PDB 3T3S), and nicotine (PDB 4EJG) have
been reported and were used for these studies. Structures of P450 2A6
3
3
(
1:1, v/v). After centrifugation, the lower organic layer was recovered
3
7
36
and the extraction was repeated. The extracts were combined and
concentrated under a nitrogen stream. HPLC separation was done
with a JASCO system (Tokyo, Japan) equipped with a Wakopack Navi
bound to coumarin (PDB 1Z10), pilocarpine (PDB 3T3R), and
nicotine (PDB 4EJJ) have also been reported. Simulations were
carried out after removing each ligand from these P450 structures
3
4
C
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Figure 4. Oxidation of naphthalene by P450s 2A6 (A) and 2A13 (B) by HPLC analysis. Standards 1-naphthol (C) and 2-naphthol (D) were also
analyzed. Formation of 1-naphthol from naphthalene by P450s 2A6 and 2A13 is shown as “a”. An asterisk (*) indicates minor peaks for products
that were not present in the absence of an NADPH-generating system.
Figure 5. Oxidation of phenanthrene (Figure 6A−G) and 2EPh (Figure 6H−N) by P450s 1A1 (A, H), 1A2 (B, I), 1B1 (C, J), 2A6 (D, K), 2A13 (E,
L), 2C9 (F, M), and 3A4 (G, N). Standard incubation conditions were used for analysis by HPLC, and duplicate determinations for each incubation
showed that very similar products were formed for these chemicals.
using the MMFF94x force field described in MOE software (ver.
obtained with these three chemicals were determined to be
.35, 2.3, and 0.88 μM, respectively, and the ΔA values
2
3−25
2
015.10, Chemical Computing Group, Montreal, Canada).
0
max
Ligand-interaction energies (U values) were obtained by use of the
ASEdock program in MOE. Lower U values indicate a stronger
interaction between a chemical and the enzyme.
Kinetic Analysis. Kinetic parameters were estimated by nonlinear
regression analysis using KaleidaGraph (Synergy Software, Reading,
PA) or GraphPad Prism (GraphPad Software, La Jolla, CA).
obtained were 0.043, 0.063, and 0.045, respectively (Figure 2).
Naphthalene and its six derivatives, phenanthrene and its six
derivatives, and biphenyl and its nine derivatives were also
compared for their abilities (ΔA /K ratio) to induce spectral
max
s
changes with P450 2A6 as well as P450 2A13 (Figure 3). It
should be pointed out that the scale for the ΔA /K ratio on
max
s
the x axis is different for P450s 2A13 and 2A6, indicating that
the former enzyme showed higher spectral changes than the
latter. 2EN induced Type I binding spectra with P450 2A13 to
a much greater extent than the parent naphthalene, and the
ΔA /K ratio for the interaction of 2EN with P450 2A13 was
RESULTS
■
Spectral Interactions of Naphthalene, Phenanthrene,
Biphenyl, and Their Derivatives with P450s 2A13 and
A6. As has been reported previously, naphthalene,
phenanthrene, and biphenyl interact with P450 2A13, inducing
Type I binding spectral changes (Figure 2). The K values
2
3
2
max
s
about 10-fold greater than that with P450 2A6 (Figure 3).
2NPE, 3EPh, and 4BPE were also found to show higher
s
D
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Figure 6. Oxidation of biphenyl by P450 2A13 by HPLC analysis with UV detection (A−C) and fluorescence detection (F−H). HPLC
chromatograms of incubations of biphenyl (50 μM) with P450 2A13 at 0.025 μM (A, F), 0.05 μM (B, G), and 0.1 μM (C, H) and chromatograms of
standard 2-hydroxybiphenyl (2.5 nmol; D, I) and standard 4-hydroxybiphenyl (E, J). Detection was by UV absorbance (A−E) or fluorescence
intensity (F−J).
intensities of interaction with P450 2A13 than parent
compounds naphthalene, phenanthrene, and biphenyl. 4BPE
was also found to induce spectral changes with P450 2A6 at a
higher level than biphenyl.
the major peak (*) seems to be similar in nature to 9-
hydroxyphenanthrene.
In order to compare the products formed for the metabolism
of phenanthrene and 2EPh, we determined their activities with
seven human P450 enzymes, P450s 1A1, 1A2, 1B1, 2A6, 2A13,
Oxidation of Naphthalene, Phenanthrene, and Bi-
phenyl by Human P450 Enzymes. Oxidation of naph-
thalene was determined with P450s 2A13 and 2A6 according to
2
C9, and 3A4 (Figure 5). The results show that P450 2A13 is
the most active at oxidizing phenanthrene (Figure 5E) and
EPh (Figure 5L) to 9-hydroxyphenanthrene and metabolite a,
13
2
the method of Fukami et al. P450s 2A13 and 2A6 produced
one major peak that corresponded to the 1-naphthol standard,
but not 2-naphthol, in the retention time on HPLC
chromatograms, with P450 2A13 showing more activity than
P450 2A6 (Figure 4). Since we did not extract naphthalene and
its metabolites and concentrate them for HPLC analysis due to
their volatile nature, it is not known whether the small peaks
seen in the chromatograms are due to 2-naphthol and other
metabolites.
respectively, followed by P450 2A6 (Figure 5D,K). Other P450
enzymes examinedP450s 1A1, 1A2, and 1B1.3were active
at producing 9-hydroxyphenanthrene from phenanthrene, and
P450s 1A2 and 1B1 oxidized 2EPh at lower levels than P450s
2
A13 and 2A6.
P450 2A13 oxidized biphenyl to 2- and/or 4-hydroxybi-
phenyl on analysis with HPLC (Figure 6). Our HPLC assay did
not separate 2- and 4-hydroxylated biphenyl products. It should
also be noted that both standards 2- and 4-hydroxybiphenyl
produced two peaks, as in the case of 9-hydroxyphenanthrene
as described above. Increasing the P450 concentration from
P450 2A13 was also active in oxidizing phenanthrene to 9-
hydroxyphenanthrene (Supporting Information Figure S1).
The 9-hydroxyphenanthrene standard and a metabolite
produced by P450 2A13 had similar, profiles showing two
peaks on HPLC chromatograms (it is not known at present
why two peaks appear for 9-hydroxyphenanthrene or if it arises
by an experimental artifact possible to detector saturation).
When zooming in on the x-axis, several minor peaks appeared
only in the presence of an NADPH-generating system with
P450 2A13 (Supporting Information Figure S1b). We also
found very similar, but different, chromatographic profiles of
product formation for the metabolism of 2EPh and
phenanthrene by P450 2A13 (Supporting Information Figure
S1C,D,c,d). We do not have a standard 2EPh metabolite, but
0
.025 to 0.1 μM caused an increase in the formation of
metabolite(s) (both with UV and fluorescence detection).
P450s 1A1, 1A2, 1B1, 2A6, 2A13, 2C9, and 3A4 were
compared for their ability to oxidize naphthalene, phenan-
threne, and biphenyl (P450 1A1 was measured in reconstituted
monooxygenase systems containing L-α-dilauroyl-sn-glycero-3-
phosphocholine and NADPH-P450 reductase as described in
the Experimental Procedures). P450 2A13 was found to be the
most active enzyme at oxidizing these chemicals, followed by
P450 2A6 (Table 1). With the other P450s examined,
naphthalene hydroxylation activity was detected with P450s
1A2, 1B1, and 3A4, in that order, but it was not detected with
E
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Table 1. Oxidation of Naphthalene, Phenanthrene, and
Biphenyl by Human P450 Enzymes
NADPH-generating system in the reaction mixture, although
detailed examinations to identify these products were not done
a
(
Figure 7). LC-MS analysis suggested the formation of
b
b
b
naphthalene
phenanthrene
biphenyl
oxygenated products of 2EPh, 2NPE, and 3EPh (results not
shown).
c
c
c
P450
1-naphthol
9-OH-phenanthrene
2- and 4-OH-biphenyl
(
nmol/min/nmol P450)
We further examined the oxidation of 21 substituted
derivatives, as well as naphthalene, phenanthrene, and biphenyl,
by P450 2A13 and compared the spectral binding constants
1A1
1A2
1B1
2A6
2A13
2C9
3A4
<0.01
0.33 ± 0.034
0.23 ± 0.024
0.02 ± 0.01
1.69 ± 0.22
3.14 ± 0.35
<0.01
0.29 ± 0.033
0.24 ± 0.027
<0.1
0.91 ± 0.11
0.41 ± 0.062
2.6 ± 0.44
6.1 ± 0.88
<0.01
(K ) of these chemicals with the enzyme (Table 2). P450 2A6
s
2.3 ± 0.31
3.1 ± 0.19
<0.1
was also used to determine the oxidation of some of the
derivatives as well as for a comparison of its spectral binding
constants with these chemicals. P450 2A13 oxidized 2EN,
1NEPE, 2NPE, 2EPh, 3EPh, 2PPh, 3PPh, 2BPE, and 4BPE as
well as the three parent compounds. Product formation was not
detected for the other chemicals with P450 2A13 in our HPLC
assay. P450 2A6 oxidized 2EN, 2EPh, 3EPh, and 4BPE as well
as the parent compounds. However, product formation (peak
height of the products) was always lower in the case of P450
0.04 ± 0.013
0.05 ± 0.02
<0.1
a
Oxidations of naphthalene, phenanthrene, and biphenyl by P450
enzymes were determined as described in the Experimental
Procedures. Data shown are the mean of three or four samples
determined ± SD. Substrate used. Product measured.
b
c
2
A6 compared to that with P450 2A13 (results not shown).
Molecular Interactions of the Chemicals Studied with
P450s 1A1 and 2C9. Phenanthrene 9-hydroxylation activity was
detected with P450s 1A1, 1A2, 3A4, and 1B1.3 but not P450
2
1
C9. Oxidation of biphenyl was detected with P450s 1A1 and
A2 but not with P450s 1B1, 2C9, and 3A4.
Oxidation of Various Substituted Derivatives of
P450s 2A13 and 2A6. Molecular docking analyses of these
chemicals with P450 2A13 were performed using reported
structures of P450 2A13 (4EJH), 2A13 (2P85), 2A13
36 34
34
35
Naphthalene, Phenanthrene, and Biphenyl by P450s
A13 and 2A6. Since our results suggested that P450s 2A13
(3T3S), and 2A13 (4EJG) bound to NNK, indole,
pilocarpine, and nicotine, respectively. Since all biological
units in the crystal structures of P450 2A13 4EJH, 2P85, and
4EJG are reported as octamers and P450 2A13 3T3S, as a
hexamer, we selected one default biological unit for docking
analysis. Docking analysis was also carried out with reported
2
and 2A6 are the major enzymes involved in the oxidation of
naphthalene, phenanthrene, and biphenyl, we extended our
study to include the metabolism of a number of substituted
derivatives of these three compounds by these enzymes. We
first evaluated the oxidation of six acetylenic derivatives, 2EN,
36
37
structures of P450 2A6 (1Z10), 2A6 (3T3R), and 2A6
34
2
NPE, 3EPh, 2PPh, 3PPh, and 4BMPE, by P450 2A13 in the
(4EJJ). Before performing simulation studies, the ligands
were removed from the monomer structures of P450 2A13, and
these ligand-free structures were used for docking analysis
(Supporting Information Figure S2). We first determined
absence and presence of an NADPH-generating system (Figure
7
2
). These six chemicals were found to be metabolized by P450
A13 to product(s) that were not present in the absence of an
Figure 7. Metabolism of 2EN (A, B), 2NPE (C, D), 3EPh (E, F), 2PPh (G, H), 3PPh (I, J), and 4BPE (K, L) by P450 2A13 in the absence (A, C, E,
G, I, and K) and presence (B, D, F, H, J, and L) of an NADPH-generating system, using HPLC analysis. Possible formation of metabolite(s) from
these chemicals is denoted with an asterisk (*) in the figure.
F
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Table 2. Spectral Binding Constants and Oxidation Results Observed for the Studied Chemicals with P450s 2A13 and 2A6
P450 2A13
P450 2A6
a
b
a
b
chemical
Type I spectra Ks (μM)
metabolism (HPLC)
Type I spectra Ks (μM)
metabolism (HPLC)
naphthalene
0.35 ± 0.02
0.080 ± 0.04
13.3 ± 3.5
1.1 ± 0.1
0.42 ± 0.10
4.9 ± 0.6
2.7 ± 0.6
2.3 ± 0.2
1.1 ± 0.3
0.45 ± 0.08
0.95 ± 0.08
3.9 ± 1.2
1.2 ± 0.4
6.4 ± 1.0
0.88 ± 0.16
1.9 ± 0.2
28 ± 12
yes
2.9 ± 0.4
1.0 ± 0.1
9.3 ± 1.8
11.5 ± 2.3
33 ± 9
yes
2
1
1
2
2
2
EN
yes
yes
NMPE
NEPE
NPE
not detected
yes
not done
not done
not done
not done
not done
yes
yes
NMPE
NEPE
not detected
not detected
yes
21 ± 0
19 ± 6
phenanthrene
2.8 ± 0.4
3.3 ± 0.4
3.3 ± 0.9
ND (10 μM)
1.6 ± 0.2
ND (10 μM)
13 ± 3
2.7 ± 0.5
5.4 ± 0.5
7.0 ± 1.9
12 ± 2
15 ± 2
0.75 ± 0.16
17 ± 3
43 ± 33
32 ± 9
2.6 ± 0.8
2
3
9
2
3
9
EPh
EPh
EPh
PPh
PPh
PPh
yes
yes
yes
yes
not detected
yes
not done
yes
yes
not done
not done
yes
not detected
yes
biphenyl
4
4
4
2
4
2
4
2
4
EB
not detected
not detected
not detected
yes
not detected
not done
not done
not done
yes
PB
BuB
3.4 ± 1.1
15.4 ± 1.7
0.48 ± 0.06
1.7 ± 0.3
66 ± 19
BPE
BPE
yes
BMPE
BMPE
2BDPE
4BDPE
not detected
not detected
not detected
not detected
not done
not done
not done
not detected
11 ± 6
6.5 ± 1.6
a
b
Data reported are the mean with SE. HPLC analysis was used for monitoring metabolism. When peak(s) were detected only in the presence of an
NADPH-generating system as described in Figure 8, metabolism results were marked as “yes”. When no metabolite peaks were detected under these
assay conditions, results were marked as “not detected”. Several of the chemicals were not examined with P450 2A6 (marked as “not done”).
Figure 8. Correlations of ligand-interaction energies (U values) using molecular docking with P450s 2A13 4EJG (nicotine-type) and 4EJH (NNK-
type) with 24 chemicals used in this study and NNK, indole, pilocarpine, nicotine, and coumarin. The latter five chemicals are indicated with blue
squares, and naphthalene, phenanthrene, and biphenyl are marked with red squares (A). Effects of spectral binding intensities (ΔA /K ratio) on
max
s
ligand binding energies (U values) using P450 2A13 4EJG (nicotine-type) (B).
optimal U values (ligand-interaction energy) (on analysis with
Information Figure S2), and that of coumarin was −34.4
results not shown).
There was a good relationship between the U values of P450
A13 4EJG (nicotine-type) and 4EJH (NNK-type) with these
chemicals (r = 0.79, p < 0.01), and we found that the parent
(
MMFF94x force field) using P450 4EJG and found that U
values for nicotine, naphthalene, phenanthrene, and biphenyl
2
were −35.2, −24.3, −29.2, and −25.7, respectively (Supporting
G
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Figure 9. Correlation of ligand-interaction energy obtained with P450 2A13 4EJG (nicotine-type) and P450 2A13 2P85 (indole-type) (A), P450
2
A13 4EJH (NNK-type) and P450 2A6 1Z10 (coumarin-type) (B), P450 2A13 4EJH (NNK-type) and P450 2A6 4EJJ (nicotine-type) (C), P450
2
A13 4EJG (nicotine-type) and P450 2A6 4EJJ (nicotine-type) (D), P450 2A6 1Z10 (coumarin-type) and P450 2A6 4EJJ (nicotine-type) (E), and
P450 2A6 4EJJ (nicotine-type) and P450 2A6 3T3R (pilocarpine-type) (F).
Figure 10. Correlation of ligand-interaction energy obtained between P450 2A13 3T3S (pilocarpine-type) and P450 2A6 3T3R (pilocarpine-type)
(
A), P450 2A6 1Z10 (coumarin-type) (B), and P450 2A6 4EJJ (nicotine-type) (C).
compounds naphthalene, phenanthrene, biphenyl, and coumar-
in had U values comparable to those of NNK, indole,
pilocarpine, nicotine, and coumarin (Figure 8). Compounds
that were oxidized by P450 2A13 (indicated by HPLC analysis)
had small U values under these assay conditions (Figure 8A).
When spectral changes (ΔA /K values) and U values (using
bound to pilocarpine, nicotine, and coumarin, respectively, was
assessed with all of the chemicals used in this study as well as
with NNK, indole, pilocarpine, nicotine, and coumarin
(Supporting Information Table S1). As in the comparison
between the U values of P450 2A13 4EJG (nicotine-type) and
4EJH (NNK-type) shown above, there were also good
corralations for combinations of the U values obtained with
these chemicals and the parent compounds, naphthalene,
phenanthrene, and biphenyl, and coumarin (marked in red)
had similar U values comparable to those of NNK, indole,
pilocarpine, nicotine, and coumarin (marked in blue) (Figure
9). However, we found relatively low correlation coefficients for
the comparison of U values of P450 2A13 3T3S bound to
pilocarpine and P450 2A6 3T3R (r = 0.37), 1Z10 (r = 0.52),
and 4EJJ (r = 0.36) bound to pilocarpine, coumarin, and
max
s
P450 2A13 4EJG for docking) were compared, some
relationship was found between these values when they were
analyzed with logarithmic curve fitting (Cricket Graph) (Figure
8
B). However, we did not find any positive correlation when U
values (with P450 2A13 4EJG) and spectral binding constants
(
K values) were compared.
A correlation of the U values using P450 2A13 4EJH, 2P85,
s
3T3S, and 4EJG bound to NNK, indole, pilocarpine, and
nicotine, respectively, and P450 2A6 3T3R, 4EJJ, and 1Z10
H
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nicotine (Figure 10), indicating that the crystal structure of
P450 2A13 with pilocarpine may be different from that with
other compounds. 44BDPE and 4BuB and some other
chemicals in structures with P450 2A6 were far from the
regression line.
acetylenic derivatives have been previously synthesized to study
the mechanisms of the inhibition of P450 enzymes, particularly
27−31,38
P450 1 and 2 family enzymes.
Our previous studies
indicated that most of these substituted chemicals, as well as
their parent compounds naphthalene, phenanthrene, and
biphenyl, are able to inhibit 7-ethoxyresorufin O-deethylation
catalyzed by P450 1B1 and coumarin 7-hydroxylation by P450s
DISCUSSION
■
23,26
2
A13 and 2A6.
In addition, 2EN has been reported to be a
The present studies show that P450 2A13 plays very important
roles in oxidizing naphthalene, phenanthrene, and biphenyl and
several of their substituted derivatives in humans and that P450
mechanism-based inhibitor of P450s 2B1, 2B4, 2B6, and
39−41
2
B11,
and our previous findings indicated that 2EN
inhibited P450 2A13- and 2A6-catalyzed coumarin 7-hydrox-
ylation with IC50 values of 1.8 and 8.8 μM, respectively. These
results suggest that the ability of some of these compounds to
be metabolized by P450 enzymes may be influenced by their
inhibition of the enzymes.
Our molecular docking simulation studies indicated that the
ligand-binding energies (U values) are correlated for all of the
2
A6 also participates in metabolizing several of these chemicals.
Other human P450s, including P450s 1A1, 1A2, 1B1, 2C9, and
A4, used in this study had lower activities at oxidizing
naphthalene, phenanthrene, and biphenyl than P450s 2A13 and
A6. Turnover rates (nmol of product formed/min/nmol
3
2
P450) of the oxidation of naphthalene, phenanthrene, and
biphenyl to 1-naphthol, 9-hydroxyphenanthrene, and 2- and/or
−1
2
4 chemicals examined when comparing the crystal structures
of P450 2A13 4EJH (NNK ligand) and P450 2A13 4EJG
nicotine ligand) (r = 0.79). Interestingly, the plotted positions
4
-hydroxybiphenyl by P450 2A13 were 6.1, 3.1, and 3.2 min ,
respectively, and catalytic activities by P450 2A6 were always
lower than those by P450 2A13. Naphthalene, phenanthrene,
and biphenyl induced Type I binding spectra with P450 2A13,
(
in Figure 8A for naphthalene, phenanthrene, and biphenyl were
at relatively similar locations to those of NNK, indole,
pilocarpine, nicotine, and coumarin, which are typical ligands
with K values of 0.35, 2.3, and 0.88 μM, respectively, and these
s
Ks values were lower than those (2.9, 2.8, and 2.7 μM,
respectively) with P450 2A6, suggesting possible relationships
between the spectral binding intensities and metabolism rates
of these chemicals by P450s 2A13 and 2A6. It should be
mentioned that we did not determine the formation of epoxide
intermediates during the metabolism of the above three
chemicals by P450 enzymes in this study, although there are
reports suggesting the production of epoxides from these
34−37
for P450 2A13.
U values were compared using P450 2A13 4EJH (NNK-type),
P85 (indole-type), 3T3S (pilocarpine-type), and 4EJG
Good correlations were also found when
2
(
(
nicotine-type), P450 2A6 3T3R (pilocarpine-type), 4EJJ
nicotine-type), and 1A10 (coumarin-type) with the chemicals
used in this study. These results indicated that the structure of
the substrate binding pocket of P450 2A6 (reported as
cocrystals with coumarin, nicotine, or pilocarpine) is almost
the same as that of P450 2A13 with NNK, nicotine, or indole.
However, there were some exceptions, with relatively low
correlation coefficients when comparing U values of P450 2A13
3T3S bound to pilocarpine and P450 2A6 3T3R (r = 0.37),
1Z10 (r = 0.52), and 4EJJ (r = 0.36) bound to pilocarpine,
coumarin, and nicotine (Figure 10). Several chemicals (e.g.,
44BDPE, 4BuB, and others) that were not good ligands for
interacting with P450s 2A13 and 2A6 and being substrates for
oxidation by these enzymes are likely to cause such weak
correlations. In addition, some other compounds (e.g 4BuB,
2BMPE, 22BDPE, and 44BDPE) that were not oxidized by
P450 2A13 showed higher ligand-binding energies on analysis
using reported P450 2A13 4EJH, 2P85, 3T3S, and 4EJG
structures bound to NNK, indole, pilocarpine, and nicotine.
These results indicate the usefulness of molecular docking
analysis in predicting possible relationships between the
interaction of chemicals with the active sites of P450 enzymes
and their susceptibilities to be metabolized by these enzymes.
Comparison of ligand-binding energies (using P450 2A13
4EJG) and spectral binding intensities with these 24 chemicals
is of interest because correlations were observed in these cases
when logarithmic extrapolation was employed (Figure 8B).
Those compounds having higher spectral binding intensities
(e.g., 2EN, 3EPh, and 2NPE) showed lower U values, whereas
compounds such as 44BDPE, 4BuB, 22BDPE, and 2BPE
which showed lower spectral binding intensitieshad higher U
values. However, it should be noted that we could not detect a
10,12,16,20,21
chemicals by P450 enzymes in laboratory animals.
Other naphthalene, phenanthrene, and biphenyl derivatives
were also analyzed for their spectral binding abilities with P450s
2
A13 and 2A6 as well as for their oxidation by these enzymes.
The results showed that compounds such as 2EN, 1NEPE,
NPE, 2EPh, 3EPh, 2PPh, 3PPh, and 4BPE, whose spectral
binding constants (K value) and intensities (ΔA /K ratio)
2
s
max
s
with P450 2A13 were comparable with those of parent
compounds naphthalene, phenanthrene, and biphenyl, were
found to be oxidized by this enzyme similarly in the presence
and absence of an NADPH-generating system. We also found
the formation of oxygenated products of 2EPh, 1NEPE, 2NPE,
and 3EPh by P450 2A13 on analysis with LC-MS. In a similar
way, 2EN, 2EPh, 3EPh, 2PPh, and 4BPE (having spectral
binding intensities with P450 2A6 comparable to those of the
parent compounds) were oxidized by this enzyme, although
product formation (peak height of the products) was always
lower for P450 2A6 than for P450 2A13, as in the cases of
naphthalene, phenanthrene, and biphenyl. P450 2A13 has 94%
amino acid sequence similarity to P450 2A6, and the volume of
3
the active site cavity of P450 2A13 (307 Å ) is known to be
larger than that of P450 2A6 (260 Å ).
3
34−37
The higher
catalytic activity of P450 2A13 compared to that of P450 2A6
toward these chemicals may be due to the size and shape of the
substrate binding pocket in the active sites of these P450s.
In this study, it was also found that there were exceptions to
the relationships observed between the spectral binding
intensities and catalytic activities for several chemicals with
P450s 2A13 and 2A6 (Table 2). For example, 2NMPE, 2NEPE,
positive correlation when spectral binding constants (K ) were
s
9
2
EPh, 9PPh, 4EB, 4BuB, 2BMPE, and 44BDPE (for P450
used for comparisons with U values. It is necessary to perform
more studies to establish the relationships between biological
responses and molecular docking results to understand the
selectivity of P450 reactions.
A13) and 4EB and 44BDPE (for P450 2A6), whose K values
s
were ≤10 μM, were not found to be oxidized by these P450
enzymes under our standard assay conditions. Most of these
I
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Chemical Research in Toxicology
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Human P450s 2A13 and 2A6 are known to catalyze the
activation and detoxication of environmental carcinogens such
as tobacco-related nitrosamines (e.g., NNK and N-nitroso-
nornicotine) and to metabolize different kinds of chemicals
including coumarin, nicotine, phenacetin, naphthalene, 4-
biphenyl metabolite(s), but P450s 1B1.1, 1B1.3, 2C9, and 3A4
did not.
In conclusion, our present study shows that P450s 2A13 and
2A6 are important enzymes at oxidizing naphthalene,
phenanthrene, biphenyl, and several of their alkynyl derivatives.
Other human P450 enzymesincluding P450s 1A1, 1A2, 1B1,
2C9, and 3A4had some role in several oxidation pathways of
these chemicals. Molecular docking analysis showed correla-
tions between ligand-interaction energies (U values) for these
13,35,42−46
aminobiphenyl, and styrene.
mainly in the respiratory tract, whereas P450 2A6 is found
P450 2A13 is expressed
4
7−49
primarily in the liver.
A13 plays a more significant role than P450 2A6 in interacting
with and metabolizing diverse environmental chemicals
We have previously shown that P450
2
2
4 chemicals with the active sites of P450s 2A13 and 2A6 and
2
3,25
the susceptibilities of these chemicals to be oxidized by the
enzymes. The results also support the usefulness of molecular
docking analysis in understanding the basis of molecular
interaction of xenobiotic chemicals with active sites of P450
proteins and possibly other enzymes.
including PAHs.
Our past and present studies also support
the importance of P450s 2A13 and 2A6 in metabolizing
40
acenaphthene and acenaphthylene as well as pyrene, 1-
50,51
hydroxypyrene, 1-nitropyrene, and 1-acethylpyrene,
and in
the present study, their importance was also shown for
metabolizing naphthalene, phenanthrene, biphenyl, and their
alkynyl derivatives.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.chemres-
tox.6b00083.
■
*
S
A role for P450 2A13 in the hydroxylation of naphthalene
13
was reported previously by Fukami et al., who showed that
naphthalene is oxidized by this enzyme to form a major
metabolite, 1-naphthol, and a minor metabolite, 2-naphthol,
through the suggested formation of naphthalene 1,2-epoxide.
They also showed that these metabolic activities of P450 2A13
were higher than those observed with P450s 1A1 and 1A2. Cho
Correlation coefficients (r values) in ligand-interaction
energies (U values) using reported structures of P450
2A13 and 2A6; oxidation of phenanthrene and 2EPh by
P450 2A13 in the absence and presence of an NADPH-
generating system (HPLC with UV detection); and
docking simulation of the interactions of nicotine,
naphthalene, phenanthrene, and biphenyl with P450
1
2
et al. also reported the in vitro metabolism of naphthalene by
human liver microsomal P450 enzymes and showed that several
P450 enzymesincluding P450s 1A2, 2A6, and 2B6catalyze
the formation of 1-naphthol and 2-naphthol on analysis with
HPLC, but they did not examine metabolism by P450 2A13.
Naphthalene has been shown to be bioactivated by mouse P450
2
A13 (4EJG) (PDF)
2
F2, goat P450 2F3, and rat P450 2F4 to lung toxicants in these
AUTHOR INFORMATION
Corresponding Authors
*(T.S.) Telephone: 72-463-5326. Fax: 72-463-5326. E-mail: t.
shimada@vet.osakafu-u.ac.jp.
■
9
,11
species;
however, the roles of human P450 2F1 (which is
reported to be present in the lung and other tissues) in the
metabolic activation of naphthalene and other chemicals
including styrene, 3-methylindole, benzene, and trichloro-
ethylene are not known at present due to difficulties with
*(S.T.) Telephone: 72-463-5326. Fax: 72-463-5326. E-mail:
takenaka@vet.osakafu-u.ac.jp.
*(H.Y.) Telephone: 42-721-1406. Fax: 42-721-1406. E-mail:
hyamazak@ac.shoyaku.ac.jp.
*(F.P.G.) Telephone: 615-322-2261. Fax: 615-322-4349. E-
mail: f.guengerich@vanderbilt.edu.
*(M.K.) Telephone: 72-463-5326. Fax: 72-463-5326. E-mail:
komori@vet.osakafu-u.ac.jp.
52
expressing P450 2F1 in heterogeneous systems such as E. coli.
Mouse P450 2A5 has been reported to play important roles in
1
1
olfactory mucosal toxicity but not in lung toxicity.
Urinary hydroxylated metabolites of phenanthrene have been
used as biomarkers to determine exposure of humans to
environmental PAHs in gasoline, diesel fuel, tobacco smoke,
5
3−55
and diet.
Several metabolitesincluding 1-, 4-, and 9-
Funding
hydroxyphenanthrenehave been identified in human
This work was supported in part by grants from the Ministry of
Education, Science, and Culture of Japan, the Ministry of
Health and Welfare of Japan (T.S., S.T., K.K., N.M., H.Y.,
M.K.), NIH grant S06 GM08008 (M.K.F.), DOE grant DE-
FC26-00NT40843 (M.K.F.), and NIH grants R37 CA090426
and P30 ES000267 (F.P.G.).
5
3−55
urine,
and in vitro studies have suggested that a number
of P450 enzymes are involved in the formation of several
hydroxylated and dihydrodiol metabolites of phenanthrene in
1
8,56
humans.
Our present results support the conclusion that 9-
hydroxyphenanthrene is a major metabolite resulting from the
incubation of phenanthrene with P450s 2A13 and 2A6.
Little is known about the metabolism of biphenyl by human
Notes
The authors declare no competing financial interest.
19
P450 enzymes. Creaven et al. reported that liver microsomes
from several animal species including rats, mice, rabbits, and
other species produce more 4-hydroxybiphenyl than 2-
hydroxybiphenyl from biphenyl. Other studies have suggested
that 3-hydroxybiphenyl (as well as 2- and 4-hydroxybiphenyl) is
formed with liver microsomes from rats, hamsters, mice, and
rabbits and that the major metabolite is 4-hydroxybiphenyl in
ABBREVIATIONS
■
P450, cytochrome P450; PAHs, polycyclic aromatic hydro-
carbons; 2EN, 2-ethynylnaphthalene; NNK, 4-(methylnitrosa-
mino)-1-(3-pyridyl)-1-butanone; 1NMPE, 1-naphthalene
methyl propargyl ether; 1NEPE, 1-naphthalene ethyl propargyl
ether; 2NPE, 2-naphthalene propargyl ether; 2NMPE, 2-
naphthalene methyl propargyl ether; 2NEPE, 2-naphthalene
ethyl propargyl ether; 2EPh, 2-ethynylphenanthrene; 3EPh, 3-
ethynylphenanthrene; 9EPh, 9-ethynylphenanthrene; 2PPh, 2-
(1-propynyl)phenanthrene; 3PPh, 3-(1-propynyl)-
20,21
all animal species examined.
Our present studies identified
2
2
- and/or 4-hydroxybiphenyl in incubations of human P450s
A13 and 2A6 with biphenyl, but the two products were not
separated in this study. Human P450s 1A1 and 1A2 produced
J
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Chemical Research in Toxicology
Article
phenanthrene; 9PPh, 9-(1-propynyl)phenanthrene; 4EB, 4-
ethynylbiphenyl; 4PB, 4-propynylbiphenyl; 4BuB, 4-butynylbi-
phenyl; 2BPE, 2-biphenyl propargyl ether; 4BPE, 4-biphenyl
propargyl ether; 2BMPE, 2-biphenyl methyl propargyl ether;
dihydrodihydroxy compounds and related glucosiduronic acids.
Biochem. J. 84, 571−582.
(
17) Chaturapit, S., and Holder, G. M. (1978) Studies on the hepatic
14
microsomal metabolism of ( C) phenanthrene. Biochem. Pharmacol.
7, 1865−1871.
18) Shou, M., Korzekwa, K. R., Krausz, K. W., Crespi, C. L.,
2
(
4
BMPE, 4-biphenyl methyl propargyl ether; 22BDPE, 2,2′-
biphenyl dipropargyl ether; 44BDPE, 4,4′-biphenyl dipropargyl
Gonzalez, F. J., and Gelboin, H. V. (1994) Regio- and stereo-selective
metabolism of phenanthrene by twelve cDNA-expressed human,
rodent, and rabbit cytochromes P-450. Cancer Lett. 83, 305−313.
(19) Creaven, P. J., Parke, D. V., and Williams, R. T. (1965) A
fluorimetric study of the hydroxylation of biphenyl in vitro by liver
preparations of various species. Biochem. J. 96, 879−885.
ether
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