Metabolism of benzo[a]pyrene to trans-7,8-dihydroxy-7,8- dihydrobenzo[a]pyrene by recombinant human cytochrome P450 1B1 and purified liver epoxide hydrolase

Article abstract of DOI:10.1021/tx990028s

Recombinant human enzymes expressed in membranes obtained from Escherichia coli transformed with cytochrome P450 (P450) and NADPH-P450 reductase cDNAs were used to identify the human P450 enzymes that are most active in catalyzing the oxidative transformation of benzo[a]pyrene in vitro. Activation of benzo[a]pyrene to genotoxic products that cause induction of umu gene expression in Salmonella typhimurium NM2009 by P450 1A1 and P450 1B1 enzymes was found to be enhanced by inclusion of purified epoxide hydrolase (isolated from rat or human livers) with the reaction mixture. High- performance liquid chromatographic analysis showed that P450 1B1 catalyzed benzo[a]pyrene to trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene at level of ~3 nmol min^-1 nmol of P450^-1 only when epoxide hydrolase was present and P450 1A1 (with the hydrolase) was able to catalyze benzo[a]pyrene at one- tenth of the activity catalyzed by P450 1B1. Kinetic analysis showed that ratio of V(max) to K(m) for the formation of trans-7,8-dihydroxy-7,8- dihydrobenzo[a]pyrene in this assay system was 3.2-fold higher in CYP1B1 than in CYP1A1. Other human P450s (including P450s 1A2, 2E1, and 3A4) were found to have very low or undetectable activities toward the formation of trans- 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene. A reconstituted system containing purified P450 1B1, rabbit liver NADPH-P450 reductase, and human liver epoxide hydrolase was found to catalyze benzo[a]pyrene to trans-7,8-dihydroxy-7,8- dihydrobenzo[a]pyrene at a rate of 0.86 nmol min^-1 nmol of P450^-1; the activities were found to be largely dependent on the presence of sodium cholate in the system. These results suggest that P450 1B1 is a principal enzyme in catalyzing the oxidation of benzo[a]pyrene to trans-7,8-dihydroxy- 7,8-dihydrobenzo[a]pyrene and that the catalytic functions of P450 1B1 may determine the susceptibilities of individuals to benzo[a]-pyrene carcinogenesis.

Full text of DOI:10.1021/tx990028s

Chem. Res. Toxicol. 1999, 12, 623-629
623
Meta bolism of Ben zo[a ]p yr en e to
tr a n s-7,8-Dih yd r oxy-7,8-d ih yd r oben zo[a ]p yr en e by
Recom bin a n t Hu m a n Cytoch r om e P 450 1B1 a n d P u r ified
Liver Ep oxid e Hyd r ola se†
Tsutomu Shimada,*,‡ Elizabeth M. J . Gillam,§ Yoshimitsu Oda,‡
Fujiko Tsumura,‡ Thomas R. Sutter,| F. Peter Guengerich, and Kiyoshi Inoue‡
Osaka Prefectural Institute of Public Health, 3-69 Nakamichi 1-chome, Higashinari-ku,
Osaka 537, J apan, Department of Physiology and Pharmacology, The University of Queensland,
St. Lucia, Queensland, Australia 4072, J ohns Hopkins School of Hygiene and Public Health,
Baltimore, Maryland 21205, and Department of Biochemistry and Center in Molecular Toxicology,
Vanderbilt University School of Medicine, Nashville, Tennessee 37232
Received February 22, 1999
Recombinant human enzymes expressed in membranes obtained from Escherichia coli
transformed with cytochrome P450 (P450) and NADPH-P450 reductase cDNAs were used to
identify the human P450 enzymes that are most active in catalyzing the oxidative transforma-
tion of benzo[a]pyrene in vitro. Activation of benzo[a]pyrene to genotoxic products that cause
induction of umu gene expression in Salmonella typhimurium NM2009 by P450 1A1 and P450
1B1 enzymes was found to be enhanced by inclusion of purified epoxide hydrolase (isolated
from rat or human livers) with the reaction mixture. High-performance liquid chromatographic
analysis showed that P450 1B1 catalyzed benzo[a]pyrene to trans-7,8-dihydroxy-7,8-dihy-
drobenzo[a]pyrene at level of ∼3 nmol min-1 nmol of P450-1 only when epoxide hydrolase was
present and P450 1A1 (with the hydrolase) was able to catalyze benzo[a]pyrene at one-tenth
of the activity catalyzed by P450 1B1. Kinetic analysis showed that ratio of Vmax to Km for the
formation of trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene in this assay system was 3.2-fold
higher in CYP1B1 than in CYP1A1. Other human P450s (including P450s 1A2, 2E1, and 3A4)
were found to have very low or undetectable activities toward the formation of trans-7,8-
dihydroxy-7,8-dihydrobenzo[a]pyrene. A reconstituted system containing purified P450 1B1,
rabbit liver NADPH-P450 reductase, and human liver epoxide hydrolase was found to catalyze
benzo[a]pyrene to trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene at a rate of 0.86 nmol min-1
nmol of P450-1; the activities were found to be largely dependent on the presence of sodium
cholate in the system. These results suggest that P450 1B1 is a principal enzyme in catalyzing
the oxidation of benzo[a]pyrene to trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene and that the
catalytic functions of P450 1B1 may determine the susceptibilities of individuals to benzo[a]-
pyrene carcinogenesis.
9â,10â-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (diol-ep-
oxide 1), (()-trans-7â,8R-dihydroxy-9R,10R-epoxy-7,8,9,-
10-tetrahydrobenzo[a]pyrene (diol-epoxide 2), and others
that cause initiation of cancers in mammals (1-7).
Several P450 enzymes have been reported to be involved
in key steps in the oxidation of B[a]P, namely, 7,8-
epoxidation of B[a]P and 9,10-epoxidation of B[a]P-7,8-
diol, and P450 1A1 has been demonstrated to be the most
active in these oxidations in mammals (1, 3, 8).
Recently, another P450 enzyme, P450 1B1, as well as
P450 1A1, has been reported to be highly active in
oxidizing several carcinogenic polycyclic aromatic hydro-
carbons, such as B[a]P and 7,12-dimethylbenz[a]an-
thracene, in laboratory animals and humans (9-12).
Particularly interesting is the observation that human
P450 1B1 is able to activate diverse procarcinogenic
chemicals, including (+)- and (-)-B[a]P-7,8-diols and
trans-3,4-dihydroxy-3,4-dihydro-7,12-dimethylbenz[a]an-
thracene, to genotoxic products that cause induction of
umu gene expression in the Salmonella typhimurium
In tr od u ction
Benzo[a]pyrene (B[a]P)1 has been shown to be bioac-
tivated by cytochrome P450 (P450) and epoxide hydrolase
to highly reactive metabolites (()-trans-7â,8R-dihydroxy-
† This work was supported in part by grants from the Ministry of
Education, Science, and Culture of J apan, the Ministry of Health and
Welfare of J apan, the Developmental and Creative Studies from Osaka
Prefectural Government (T.S.), by the Kathleen Cunningham Founda-
tion for Breast Cancer Research (E.M.J .G.), and by U.S. Public Health
Service Grants ES08148 and ES03819 (T.R.S.) and CA44353 and
ES00267 (F.P.G.).
* To whom correspondence should be addressed: Osaka Prefectural
Institute of Public Health, 3-69 Nakamichi 1-chome, Higashinari-ku,
Osaka 537-0025, J apan. Telephone: (+81)-6-6972-1321. Fax: (+81)-
6-6972-2393. E-mail: shimada@iph.pref.osaka.jp.
‡ Osaka Prefectural Institute of Public Health.
§ The University of Queensland.
| J ohns Hopkins School of Hygiene and Public Health.
Vanderbilt University School of Medicine.
1
Abbreviations: P450, cytochrome P450; B[a]P, benzo[a]pyrene;
B[a]P-7,8-diol, trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene; DLPC,
L-R-dilauroyl-sn-glycero-3-phosphocholine; HPLC, high-performance
liquid chromatography.
10.1021/tx990028s CCC: $18.00 © 1999 American Chemical Society
Published on Web 06/17/1999

624 Chem. Res. Toxicol., Vol. 12, No. 7, 1999
Shimada et al.
Ta ble 1. Effects of Ep oxid e Hyd r ola se on th e Meta bolic
Activa tion of Ben zo[a ]p yr en e by Recom bin a n t P 450 1A1
a n d P 450 1B1 Exp r essed in Bicistr on ic F or m a t w ith
NADP H-P 450 Red u cta se in E. coli Mem br a n esa
NM2009 strain (13-16). This enzyme has been reported
to be expressed mainly in extrahepatic organs, including
mammary glands, uterus, prostate, testis, adrenals, lung,
kidney, etc., and has been suggested to be one of the key
enzymes in understanding the basis for chemically
induced breast and lung cancers in humans (12, 13, 17-
19).
We report the results of our studies involving the
metabolism of B[a]P to B[a]P-7,8-diol using HPLC analy-
sis of metabolites and the activation of these procarcino-
gens to reactive metabolites that cause induction of umu
gene expression in S. typhimurium NM2009 by recom-
binant human P450 enzymes and purified rat or human
liver epoxide hydrolase. Recombinant enzymes in mem-
branes prepared from Escherichia coli into which human
P450 and NADPH-P450 reductase cDNAs have been
introduced and purified P450 1B1 isolated from mem-
branes of E. coli expressing only the cognate P450 were
used throughout this study.
activation of B[a]P
P450 1A1
P450 1B1
units min-1
units min-1
epoxide hydrolase nmol of P450-1
%
nmol of P450-1
%
none
rat enzyme
human enzyme
293 ( 43 (100)
537 ( 25
476 ( 61
410 ( 46 (100)
183 725 ( 42
162 829 ( 99
177
202
a
The incubation mixture contained 10 pmol of P450 1A1 or
P450 1B1 (added in the form of membranes isolated from bacteria
which coexpress NADPH-P450 reductase), an NADPH-generating
system, 50 mM potassium phosphate buffer (pH 7.4), and 5 µM
B[a]P in the presence or absence of 40 pmol of rat and human
epoxide hydrolase. The mixtures were first incubated at 37 °C for
5 min, and then bacterial (S. typhimurium NM2009) solutions
were added and the mixtures further incubated at 37 °C for 2 h.
Data represent the mean ( SD of triplicate determinations.
terminated by the addition of 500 µL of cold acetone. The
reaction mixtures containing B[a]P and its metabolites were
extracted twice with 1 mL of ethyl acetate, and the organic
layers were combined and evaporated under a nitrogen atmo-
sphere. The residues were dissolved in 500 µL of CH3OH, and
an aliquot (usually 25 µL) was used for HPLC analysis with a
LC-CCPS system (Tosoh Co., Tokyo, J apan) with an FS-8020
spectrofluorometer (Tosoh Co.). Separation was achieved with
a C18 5 µm analytical column (Mightsil RP-18, 150 mm × 4.6
mm, Kanto Chemical Co., Tokyo, J apan) equipped with a C18
5 µm guard column (Mightsil RP-18, 5 mm × 4.6 mm, Kanto
Chemical Co.). The eluent consisted of 64% CH3OH (64:36, v/v).
The flow rate was 1.0 mL/min, and the fluorimetric detection
was carried out at an excitation wavelength of 340 nm and an
emission wavelength of 402 nm. Peak areas thus obtained were
integrated with a Chromatopac Instrument (C-R6A Chromato-
pac, Shimadzu, Kyoto, J apan).
Exp er im en ta l P r oced u r es
Ca u tion : Benzo[a]pyrene and trans-7,8-dihydroxy-7,8-dihy-
drobenzo[a]pyrene are mutagenic and carcinogenic and should
be handled with care.
Ch em ica ls. (+)- and (-)-B[a]P-7,8-diols were purchased from
the National Cancer Institute Chemical Carcinogen Repository
Midwest Research Institute (Kansas City, MO). B[a]P, DLPC,
l-R-dioleoyl-sn-glycero-3-phosphocholine, phosphatidylserine, and
cholic acid were purchased from Sigma Chemical Co. (St. Louis,
MO). Cholic acid was recrystallized from hot 50% ethanol using
activated charcoal and Celite to remove impurities (20). Other
chemicals and reagents used in this study were obtained from
sources described previously or were of the highest qualities
commercially available (21, 22).
En zym es. Bacterial P450 1A1, 1A2, 1B1, 2E1, and 3A4
systems were prepared as described previously (23, 24). Briefly,
plasmids constructed to enable the expression of P450 1A1, 1A2,
1B1, 2E1, or 3A4 and human NADPH-P450 reductase (using
a single promoter) were introduced into E. coli DH5R cells by a
heat shock procedure, and the transformants were selected on
Luria-Bertani medium containing 100 µg/mL ampicillin. Bac-
teria were cultured as previously described (23), and membranes
were prepared and suspended in 10 mM Tris-HCl buffer (pH
7.4) containing 1.0 mM EDTA and 20% glycerol (v/v) (23).
Human P450 1B1 was purified from membranes of E. coli in
which human P450 1B1 plasmids (expressing only the P450)
had been introduced as described previously (23).
7-Ethoxyresorufin O-deethylation was assayed as described
previously (23, 29).
Gen otoxicity Assa ys. P450-dependent activation of B[a]P
or B[a]P-7,8-diol to reactive products that cause induction of
umu gene expression in tester strain S. typhimurium NM2009
was evaluated in systems containing P450 1A1 or P450 1B1
(added in the form of membranes isolated from E. coli which
coexpress human NADPH-P450 reductase) as described previ-
ously (13, 30). Standard incubation mixtures included P450 (10
pmol) and 5.0 µM procarcinogen in a final volume of 1.0 mL of
100 mM potassium phosphate buffer (pH 7.4) containing an
NADPH-generating system and 0.75 mL of bacterial suspension.
Incubation conditions for E. coli membrane and reconstitution
systems were as described above. The induction of umu gene
expression is presented in units of â-galactosidase activity per
minute per nanomole of P450 (31).
Rat liver epoxide hydrolase was prepared as described
elsewhere (25, 26). Human epoxide hydrolase was isolated from
liver sample HL96, using modifications of previously described
techniques (26).
Rabbit NADPH-P450 reductase was purified as described
previously (27, 28).
Oth er Assa ys. P450 was identified spectrally by the method
of Omura and Sato (32). Protein concentrations were estimated
by the method of Lowry et al. (33).
HP LC An a lysis of B[a ]P Meta bolism . Standard reaction
mixtures for the metabolism of B[a]P included P450 (10 pmol)
in bacterial membranes, rat or human epoxide hydrolase (40
pmol), and DLPC (2.5 µg) in 50 mM potassium phosphate buffer
(pH 7.4) containing an NADPH-generating system (consisting
of 0.5 mM NADP+, 5 mM glucose 6-phosphate, and 0.5 unit/
mL glucose-6-phosphate dehydrogenase) and 20 µM B[a]P
dissolved in MeOH in a final volume of 0.25 mL. The solvent
MeOH did not affect the catalytic activities under this assay
condition. In the case of purified P450 1B1 (10 pmol), NADPH-
P450 reductase (20 pmol) and 2.5 µg/mL of phospholipid mixture
(DLPC, L-R-dioleoyl-sn-glycero-3-phosphocholine, and phosphati-
dylserine, 1:1:1, w/w/w) were added for the reconstitution, and
sodium cholate (final concentration of 0.01%, w/v) was also
included to improve catalytic activities as described previously
(23). The reaction was conducted at 37 °C for 10 min and
Resu lts
Activa tion of B[a ]P or B[a ]P -7,8-d iol in S. typh -
im u r iu m NM2009. B[a]P was activated by P450 1A1 or
P450 1B1 (added in the form of membranes isolated from
E. coli cells which coexpress human NADPH-P450
reductase) to reactive metabolites that caused induction
of umu gene expression in S. typhimurium NM2009
(Table 1). In the absence of epoxide hydrolase, P450 1B1
was about 1.4-fold more active than P450 1A1 in the
activation of B[a]P. When purified epoxide hydrolases
isolated from rat or human livers were included in the
system, the activities catalyzed by both P450 1A1 and

Human P450 1B1 and Benzo[a]pyrene
Chem. Res. Toxicol., Vol. 12, No. 7, 1999 625
F igu r e 3. Metabolism of B[a]P by recombinant P450 1B1 and
human epoxide hydrolase. Standard reaction mixtures were
used except that (A) the NADPH-generating system was omitted
and (B) the epoxide hydrolase was removed from the reaction
mixture. HPLC profiles of (+)- and (-)-B[a]P-7,8-diol (4 pmol
of each) are presented. Peaks a-c correspond to unidentified
metabolites, and peak d corresponds to (+)- or (-)-B[a]P-7,8-
diol.
F igu r e 1. Effects of increasing concentrations of R-naphtho-
flavone (A) and rat epoxide hydrolase (B) on the activation of
B[a]P by recombinant P450 1B1 (expressed in bicistronic format
with NADPH-P450 reductase in E. coli membranes). The
incubation mixture (final volume of 1.0 mL) contained P450 (10
pmol), the potassium phosphate buffer (pH 7.4, 50 µmol), the
NADPH-generating system, B[a]P (5 nmol), and 0.75 mL of
bacterial suspension in the absence and presence of different
concentrations of R-naphthoflavone and rat epoxide hydrolase.
Data are means of duplicate determinations.
F igu r e 4. Effect of increasing concentrations of human epoxide
hydrolase on the metabolism of B[a]P by recombinant P450 1B1
(expressed in bicistronic format in bacterial membranes). Stan-
dard incubation mixtures were used, except that different
concentrations of epoxide hydrolase were included in the
mixture. Data are means of duplicate determinations.
F igu r e 2. Effects of increasing concentrations of a-naphtho-
flavone (A) and rat epoxide hydrolase (B) on the activation of
(-)B[a]P-7,8-diol by recombinant P450 1B1 (expressed in bicis-
tronic format with NADPH-P450 reductase in E. coli mem-
branes). Experimental conditions are the same as described in
the legend of Figure 1, except that B[a]P was replaced by (-)-
B[a]P-7,8-diol (5 nmol). Data are means of duplicate determina-
tions.
epoxide hydrolase resulted in the disappearance of any
peaks (a-d) on the HPLC chromatograms.
The extent of formation of B[a]P-7,8-diol from B[a]P
by bicistronic P450 1B1 in bacterial membranes was
found to be increased with increasing concentrations of
human epoxide hydrolase in the reaction mixture (Figure
4).
Roles of Differ en t F or m s of Hu m a n P 450 in th e
Meta bolism of B[a ]P . Five different preparations of
human enzymes (P450 1A1, 1A2, 1B1, 2E1, and 3A4) in
membranes of E. coli in which the respective P450 and
NADPH-P450 reductase cDNAs had been introduced
were used to determine the P450 enzymes that are the
most active in catalyzing B[a]P to B[a]P-7,8-diol and
other metabolites using HPLC analysis (Figure 5 and
Table 2). In all cases, 40 pmol of human epoxide hydro-
lase was included. The 7-ethoxyresorufin O-deethylation
activities catalyzed by P450 1A1, 1A2, and 1B1 are also
shown in Table 2 for reference to other systems. P450
1A1 was the most active in 7-ethoxyresorufin O-deeth-
ylation activity, followed by P450 1B1 and P450 1A2.
P450 1B1, followed by P450 1A1, was found to be active
in catalyzing the metabolism of B[a]P to B[a]P-7,8-diol
P450 1B1 for B[a]P activation in S. typhimurium NM2009
were increased by 1.6-2-fold in both systems.
R-Naphthoflavone inhibited and epoxide hydrolase
(from rat livers) stimulated the activation of P450 1B1-
dependent activation of B[a]P in S. typhimurium NM2009
in a concentration-dependent manner (Figure 1). Activa-
tion of B[a]P-7,8-diol by P450 1B1 was also inhibited by
R-naphthoflavone, whereas the activities were not af-
fected by epoxide hydrolase at all (Figure 2).
Meta bolism of B[a ]P to B[a ]P -7,8-d iol by P 450
1B1 a n d Ep oxid e Hyd r ola se. Effects of epoxide hy-
drolase (from human livers) on the metabolism of B[a]P
by P450 1B1 (expressed in bacterial membranes with
human NADPH-P450 reductase) were examined by
HPLC using fluorescence detection (Figure 3). In the
presence of human epoxide hydrolase, P450 1B1 con-
verted B[a]P to B[a]P-7,8-diol (peak d), the minor prod-
ucts, and unidentified minor metabolites (peaks a-c).
Removal of either the NADPH-generating system or

626 Chem. Res. Toxicol., Vol. 12, No. 7, 1999
Shimada et al.
F igu r e 6. Reconstitution of the metabolism of B[a]P by purified
recombinant P450 1B1 and human liver epoxide hydrolase. The
standard reaction mixture (final volume of 0.25 mL) contained
P450 1B1 (10 pmol), rabbit NADPH-P450 reductase (20 pmol),
epoxide hydrolase (40 pmol), the lipid mixture (2.5 µg), sodium
cholate (final concentration of 0.01%), the potassium phosphate
buffer (pH 7.4, 50 µmol), an NADPH-generating system, and
B[a]P (5 nmol). The effects of different concentrations of epoxide
hydrolase on the P450 1B1-dependent metabolism were exam-
ined; the amounts of epoxide hydrolase added to the mixtures
were 0 (A), 10 (B), 20 (C), and 40 pmol (D, complete system).
For panel E, the sodium cholate was not included in the
complete system. Peaks a-c correspond to unidentified metabo-
lites, and peak d corresponds to (+)- or (-)-B[a]P-7,8-diol.
F igu r e 5. Metabolism of B[a]P by recombinant P450 1A1 (A),
P450 1A2 (B), P450 1B1 (C), P450 2E1 (D), and P450 3A4 (E)
(expressed in bicistronic format in bacterial membranes) in the
presence of human epoxide hydrolase. Standard reaction mix-
tures were used, except that different recombinant P450s (10
pmol of each) were used in each case. Peaks a-c correspond to
unidentified metabolites, and peak d corresponds to (+)- or (-)-
B[a]P-7,8-diol.
Ta ble 2. 7-Eth oxyr esor u fin O-Deeth yla tion a n d
B[a ]P -7,8-d iol P r od u ct F or m a tion by Hu m a n
Recom bin a n t P 450 En zym es in th e P r esen ce of Hu m a n
Ep oxid e Hyd r ola sea
from membranes isolated from E. coli in which the P450
1B1 cDNA (but not the NADPH-P450 reductase cDNA)
had been introduced was used for reconstitution of the
metabolism of B[a]P (Figure 6). In this case, sodium
cholate (final concentration of 0.01%, w/v) was required
for the reconstitution of metabolism of B[a]P, as has been
reported by us previously for the catalysis of 7-ethoxyre-
sorufin O-deethylation activities (23, 35). It was also
found that the extent of formation of B[a]P-7,8-diol and
other unidentified metabolites was increased with the
concentrations of epoxide hydrolase in the incubation
mixtures (panels B-D). The turnover number for the
formation of B[a]P-7,8-diol by P450 1B1 in the complete
system was found to be 0.86 ( 0.11 nmol min-1 nmol of
P450-1 (n ) 3).
7-ethoxyresorufin
formation of
O-deethylation (nmol min-1
B[a]P-7,8-diol (nmol
nmol of P450-1
)
min-1 nmol of P450-1
)
P450 1A1
P450 1A2
P450 1B1
P450 2E1
P450 3A4
25.2 ( 3.2
1.1 ( 0.3
6.2 ( 1.0
ND
0.27 ( 0.05
0.02 ( 0.01
2.69 ( 0.22
<0.01
ND
<0.01
a
The incubation mixture (final volume of 1.0 mL for 7-ethoxy-
resorufin and 0.25 mL for B[a]P metabolism) in the bicistronic
system consisted of P450 (10 pmol; added in the form of E. coli
membranes also containing coexpressed NADPH-P450 reductase)
and 7-ethoxyresorufin or B[a]P (10 nmol in methanol in both cases)
in potassium phosphate buffer (pH 7.4, 50 µmol) containing the
NADPH-generating system. In the case of B[a]P metabolism,
standard incubation mixtures as described in Experimental
Procedures were used. ND means not done. Data are means (
SD of triplicate determinations.
Discu ssion
and three unidentified metabolites. P450 1A2 produced
B[a]P-7,8-diol at very low rate, but none of the metabo-
lites were detected in systems containing P450 2E1 and
P450 3A4 using these HPLC conditions. In this study,
we did not add cytochrome b5 into the reaction mixtures,
since recent studies have provided evidence suggesting
that recombinant P450 2E1 and 3A4 coexpressing NAD-
PH-P450 reductase in E. coli gave high rates for the
substrate oxidations that were studied (24, 34). The
turnover number of the formation of B[a]P-7,8-diol by
P450 1B1 was determined to be 2.7 nmol min-1 nmol of
P450-1; the value was about 10-fold higher than the
activity catalyzed by P450 1A1 in this system (Table 2).
The rate of this reaction catalyzed by P450 1A2 was
The turnover numbers for the formation of B[a]P-7,8-
diol on the incubation of B[a]P with bicistronic P450 1B1
and P450 1A1 expressed using bicistronic systems were
calculated to be about 2.7 and 0.27 nmol min-1 nmol of
P450-1, respectively. The rate achieved by P450 1A1 is
similar to the numbers reported by Kim et al. (36) and
Bauer et al. (37), who showed that recombinant P450 1A1
in baculovirus-infected cells and in yeast, respectively,
catalyzed metabolism of B[a]P to B[a]P-7,8-diol at a rate
of ∼0.6 nmol min-1 nmol of P450-1. However, Kim et al.
(36) reported that the rate of formation of B[a]P-7,8-diol
was decreased to ∼0.4 nmol min-1 nmol of P450-1 when
recombinant epoxide hydrolase (in human lymphoblast
cell microsomes) is included in the system. They also
reported that rates of formation of B[a]P-7,8-diol by two
preparations of recombinant P450 1B1 (expressed in
baculovirus-infected cells or yeast) were 0.17 and 0.31
nmol min-1 nmol of P450-1 even when recombinant
epoxide hydrolase is included in the system. In our work
presented here, the formation of B[a]P-7,8-diol from
B[a]P was detected only in the presence of purified
epoxide hydrolase in the system, and the turnover
number for B[a]P metabolism to B[a]P-7,8-diol by bicis-
∼0.02 nmol of B[a]P-7,8-diol min-1 nmol of P450-1
.
The kinetic analysis of the formation of B[a]P-7,8-diol
by P450 1A1 and P450 1B1 was carried out using
substrate concentrations between 4 and 20 µM B[a]P. Km
values for P450 1A1 and P450 1B1 were determined to
be 10 ( 5 and 17 ( 9 µM, respectively, and Vmax values
were 0.6 ( 0.1 and 3.3 ( 1.9 nmol min-1 nmol of P450-1
respectively.
,
Recon stitu tion of B[a ]P Meta bolism by P u r ified
P 450s a n d Ep oxid e Hyd r ola se. P450 1B1 purified

Human P450 1B1 and Benzo[a]pyrene
Chem. Res. Toxicol., Vol. 12, No. 7, 1999 627
tronic P450 1B1 was ∼2.7 nmol min-1 nmol of P450-1
.
tabolites, such as B[a]P-4,5-oxide, which do not require
further hydrolysis by epoxide hydrolase, to exert their
DNA-damaging activities (1, 3).
Kinetic analysis showed that ratio of Vmax to Km for the
formation of B[a]P-7,8-diol by P450 1A1 and CYP1B1 was
0.06 and 0.19 mL min-1 nmol of P450-1, respectively.
These results support the view that P450 1B1 is more
active in catalyzing B[a]P to B[a]P-7,8-diol than P450
1A1.
In the genotoxicity assay presented here, the activities
of P450 1B1-dependent activation of B[a]P-7,8-diol to
DNA-damaging products in S. typhimurium NM2009
were found to be unaffected by inclusion of epoxide
hydrolase in the reaction mixture, although R-naph-
thoflavone was effective in inhibiting the activities
(Figure 2). In this connection, the results of Glatt et al.
(49) are interesting in that the mutagenicity of a diol
epoxide of benz[a]anthracene was not decreased by rat
liver microsomal epoxide hydrolases in the Ames assay
system using S. typhimurium TA100 as a tester strain,
whereas the mutagenicity of benz[a]anthracene 5,6-oxide
was significantly inhibited by the hydrolase. The authors
have also showed that the purified rat dihydrodiol
dehydrogenase inhibits the mutagenicity of a diol epoxide
of benz[a]anthracene but not that of the 5,6-oxide (49).
In conclusion, these results indicate further that P450
1B1 is considerably important in understanding the basis
for metabolic activation of carcinogenic polycyclic aro-
matic hydrocarbons, such as B[a]P in humans. This
enzyme, along with epoxide hydrolase, catalyzes metabo-
lism of B[a]P to B[a]P-7,8-diol which has been shown to
be highly metabolized by P450 1B1 and P450 1A1 to
active metabolites that are genotoxic in the S. typhimu-
rium NM2009 strain (13, 23). The turnover number for
the formation of B[a]P-7,8-diol by P450 1B1 was calcu-
lated to be ∼2.7 nmol min-1 nmol of P450-1; the rate was
about 10-fold higher than those catalyzed by P450 1A1.
Kinetic analysis showed that ratio of Vmax to Km for the
formation of trans-7,8-dihydroxy-7,8-dihydrobenzo[a]py-
rene in this assay system was 3.2-fold higher in CYP1B1
than in CYP1A1. The reconstituted system containing
P450 1B1 also exhibited high rates for the formation of
B[a]P-7,8-diol, and we found that the reconstitution
required sodium cholate for full catalytic activities in the
metabolism of B[a]P. The relative contributions of P450s
1A1 and 1B1 will be influenced by the levels of expression
of the proteins in each tissue.
Using HPLC analysis with fluorescence detection, we
found three unidentified metabolites (a-c) as well as (+)-
or (-)-B[a]P-7,8-diol (peak d). The fluorescence intensi-
ties and retention time for (+)- and (-)-B[a]P-7,8-diol
were almost identical under the HPLC assay conditions
described here. In regard to other peaks found in the
HPLC analysis, we have obtained preliminary evidence
that peak c may be B[a]P-trans-4,5-dihydrodiol, although
the other two metabolites have not been identified yet.
Sodium cholate, as well as epoxide hydrolase, was
necessary for full catalytic activity in the metabolism of
B[a]P to B[a]P-7,8-diol and other metabolites in recon-
stituted systems containing purified P450 1B1; the
results are consistent with our previous studies which
showed that 7-ethoxyresorufin O-deethylation is en-
hanced by cholate using recombinant purified P450 1B1,
recombinant P450 1B1 in membranes of E. coli, and P450
1B1 in microsomes of yeast (23, 35). In this connection,
it is interesting to note our preliminary accounts which
showed that activities of 7-ethoxyresorufin O-deethyla-
tion by P450 1B1 in microsomes of yeast are enhanced
by addition of external (rabbit and human) NADPH-
P450 reductase to the incubation mixture, and addition
of sodium cholate in this reaction mixture causes further
increases in the activities (35). These results suggest that
interaction between P450 1B1 and NADPH-P450 re-
ductase in membranes of E. coli and in microsomes of
yeast may be enhanced in the presence of sodium cholate.
Human mammary epithelial cells have been shown to
be able to convert B[a]P and its 7,8-dihydrodiol and
dibenzo[a,l]pyrene to more polar metabolites (38, 39). The
literature suggests that P450 1B1, but not P450 1A1, is
constitutively expressed in primary cultured human
mammary stromal fibroblasts; the cells respond to dioxin
exposure (17, 40, 41). These results are of interest in
understanding the basis of mammary cancers resulting
from exposure to environmental polycyclic aromatic
hydrocarbons in humans, particularly the roles of P450
1B1 in the metabolic activation of carcinogenic polycyclic
aromatic hydrocarbons as well as in the oxidation of
estrogens (42-46).
In this study, the formation of B[a]P-7,8-diol was found
to be largely dependent on the presence of epoxide
hydrolase in the incubation mixture. The dependence of
the formation of B[a]P-7,8-diol and other diol metabolites
on epoxide hydrolase has already been reported by others
(47, 48) in systems containing human P450 1A1 and
epoxide hydrolase expressed in yeast or in human lym-
phoblastoid cell line and vaccina virus systems. The
results presented here from the umu assay for the
detection of genotoxic activation of B[a]P by P450s in S.
typhimurium NM2009 also indicate that epoxide hydro-
lase stimulated the P450 1A1- and P450 1B1-dependent
activation of B[a]P by 1.6-2-fold. However, our results
suggest that B[a]P was metabolized by P450 1A1 and
P450 1B1 to form active metabolites that cause induction
of umu gene expression in the absence of epoxide hydro-
lase. These results suggest that P450 1A1 or P450 1B1
catalyzes the activation of B[a]P to several active me-
Ack n ow led gm en t. We thank Dr. H. Yamazaki for
his helpful suggestions regarding HPLC analysis.
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