N-Nitrosobenzylmethylamine hydroxylation and coumarin 7-hydroxylation: Catalysis by rat esophageal microsomes and cytochrome P450 2A3 and 2A6 enzymes

Article abstract of DOI:10.1021/tx990128y

N-Nitrosobenzylmethylamine (NBzMA) is a potent and selective esophageal carcinogen in the rat and may be a causative agent for human esophageal cancer. This nitrosamine, like most, must be metabolically activated to exert its carcinogenic potential. NBzMA may be metabolized by P450-catalyzed methyl or methylene hydroxylation; the latter is believed to be the activation pathway. The sensitivity of the esophagus to NBzMA-induced tumorigenesis is believed to be due, at least in part, to the presence of efficient P450 catalysts in this tissue. However, while it was reported almost 20 years ago that the rat esophagus catalyzes the methylene hydroxylation of NBzMA, the P450 that catalyzes this reaction has yet to be identified. We report here that human P450 2A6 and the closely related extrahepatic rat enzyme P450 2A3 both efficiently catalyze NBzMA methylene hydroxylation, characterized as benzaldehyde formation. The catalytic efficiency of P450 2A3 in this reaction was 3-fold greater than that of P450 2A6, 7.6 (K(m) = 0.63 ± 0.18 μM and the V(max) = 4.8 nmol min^-1 nmol of P450^-1) versus 2.3 (K(m) = 6.7 ± 2.9 μM and the V(max) = 15.7 nmol min^-1 nmol of P450^-1), respectively. Both enzymes catalyzed methylene hydroxylation at least 4-fold more efficiently than methyl hydroxylation. In addition, P450 2A6, but not P450 2A3, catalyzed benzyl ring hydroxylation, generating N-(p-hydroxybenzyl)methylamine. The identity of this metabolite was confirmed by synthesis of a standard and LC/MS and LC/MS/MS analysis. P450 2A6 is an efficient coumarin 7-hydroxylase, and we report here that P450 2A3 is an equally good catalyst of this reaction (K(m) = 1.7 ± 0.41 μM and V(max) = 1.7 ± 0.08 nmol min^-1 nmol of P450^{- 1}). Rat esophageal microsomes (REM), like P450 2A3, were efficient catalysts of NBzMA methylene hydroxylation. However, in contrast to P450 2A3, the major product of this reaction was the product of benzaldehyde oxidation, benzoic acid. Antibody to the closely related mouse P450, 2A5, did not inhibit REM- catalyzed NBzMA metabolism, and most importantly, REM did not catalyze the 7- hydroxylation of coumarin. Therefore, P450 2A3 does not appear to be the P450 in the rat esophagus responsible for catalyzing the methylene hydroxylation of NBzMA.

Full text of DOI:10.1021/tx990128y

1254
Chem. Res. Toxicol. 1999, 12, 1254-1261
N-Nitr osoben zylm eth yla m in e Hyd r oxyla tion a n d
Cou m a r in 7-Hyd r oxyla tion : Ca ta lysis by Ra t Esop h a gea l
Micr osom es a n d Cytoch r om e P 450 2A3 a n d 2A6 En zym es
Linda B. von Weymarn,† Nadia D. Felicia,† Xinxin Ding,‡ and
Sharon E. Murphy*,†
Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota Cancer
Center, Minneapolis, Minnesota 55455, and Laboratory of Human Toxicology and Molecular
Epidemiology, Wadsworth Center, New York State Department of Health, Albany, New York 12201
Received J uly 13, 1999
N-Nitrosobenzylmethylamine (NBzMA) is a potent and selective esophageal carcinogen in
the rat and may be a causative agent for human esophageal cancer. This nitrosamine, like
most, must be metabolically activated to exert its carcinogenic potential. NBzMA may be
metabolized by P450-catalyzed methyl or methylene hydroxylation; the latter is believed to be
the activation pathway. The sensitivity of the esophagus to NBzMA-induced tumorigenesis is
believed to be due, at least in part, to the presence of efficient P450 catalysts in this tissue.
However, while it was reported almost 20 years ago that the rat esophagus catalyzes the
methylene hydroxylation of NBzMA, the P450 that catalyzes this reaction has yet to be
identified. We report here that human P450 2A6 and the closely related extrahepatic rat enzyme
P450 2A3 both efficiently catalyze NBzMA methylene hydroxylation, characterized as
benzaldehyde formation. The catalytic efficiency of P450 2A3 in this reaction was 3-fold greater
than that of P450 2A6, 7.6 (Km ) 0.63 ( 0.18 µM and the Vmax ) 4.8 nmol min-1 nmol of
P450-1) versus 2.3 (Km ) 6.7 ( 2.9 µM and the Vmax ) 15.7 nmol min-1 nmol of P450-1),
respectively. Both enzymes catalyzed methylene hydroxylation at least 4-fold more efficiently
than methyl hydroxylation. In addition, P450 2A6, but not P450 2A3, catalyzed benzyl ring
hydroxylation, generating N-(p-hydroxybenzyl)methylamine. The identity of this metabolite
was confirmed by synthesis of a standard and LC/MS and LC/MS/MS analysis. P450 2A6 is
an efficient coumarin 7-hydroxylase, and we report here that P450 2A3 is an equally good
catalyst of this reaction (Km ) 1.7 ( 0.41 µM and Vmax ) 1.7 ( 0.08 nmol min-1 nmol of P450-1).
Rat esophageal microsomes (REM), like P450 2A3, were efficient catalysts of NBzMA methylene
hydroxylation. However, in contrast to P450 2A3, the major product of this reaction was the
product of benzaldehyde oxidation, benzoic acid. Antibody to the closely related mouse P450,
2A5, did not inhibit REM-catalyzed NBzMA metabolism, and most importantly, REM did not
catalyze the 7-hydroxylation of coumarin. Therefore, P450 2A3 does not appear to be the P450
in the rat esophagus responsible for catalyzing the methylene hydroxylation of NBzMA.
of these nitrosamines in the esophagus has not been
identified.
In tr od u ction
A number of different nitrosamines preferentially
induce esophageal tumors in the rat (1, 2). N-Nitrosoben-
zylmethylamine (NBzMA)1 is the most potent esophageal
carcinogen that has been tested (3). NBzMA, like most
nitrosamines, requires metabolic activation to exert its
carcinogenic potential (1). Several investigators have
proposed that the esophagus contains an enzyme(s) that
catalyzes the activation of nitrosamines (4, 5). Data that
support the role of an esophageal P450 in the metabolism
of NBzMA, N′-nitrosonornicotine (NNN), and N-nitroso-
methylpentylamine (NMAA) have been reported (6-9).
However, the enzyme(s) responsible for the metabolism
The activation of NBzMA is proposed to occur by P450-
catalyzed methylene hydroxylation (6, 10, 11). The hy-
droxylated nitrosamine (Scheme 1) decomposes to benz-
aldehyde and methane diazohydroxide, which can react
with DNA to generate O6-methylguanine. Miscoding due
to this promutagenic base is believed to be the initiating
event in NBzMA-induced esophageal carcinogenesis (5,
12-14). Benzaldehyde can be further oxidized to benzoic
acid (6). Hydroxylation may also occur at the methyl
carbon, generating formaldehyde and a benzylating
agent. The latter will react with water to form benzyl
alcohol or can react with DNA. However, in NBzMA-
treated rats, DNA benzylation occurs at very low levels
in the liver, orders of magnitude lower than the level of
DNA methylation (15). DNA benzylation has not been
detected in the esophagus.
† University of Minnesota Cancer Center.
‡ New York State Department of Health.
Abbreviations: NBzMA, N-nitrosobenzylmethylamine; NNN, N′-
nitrosonornicotine; NMAA, N-nitrosomethylpentylamine; N(p-OHBz)-
MA, N-nitroso(p-hydroxybenzyl)methylamine; BV, baculovirus; REM,
rat esophagus microsomes; APCI, atmospheric-pressure chemical
ionization.
1
Consistent with tissue specific activation of NBzMA,
Labuc and Archer reported that rat esophageal mi-
10.1021/tx990128y CCC: $18.00 © 1999 American Chemical Society
Published on Web 11/18/1999

NBzMA Hydroxylation by P450 2A3 and Rat Esophagus
Chem. Res. Toxicol., Vol. 12, No. 12, 1999 1255
Sch em e 1. Hyd r oxyla tion P a th w a ys of NBzMA Meta bolism
Ch em ica ls. NBzMA was obtained from the NCI Chemical
Carcinogen Repository (Midwest Research Institute, Kansas
City, MO). [3H-benzyl]NBzMA (1.8 Ci/mmol) was a gift from L.
Peterson (University of Minnesota Cancer Center). The tritium
is meta to the methylene group of NBzMA (15). NBzMA
metabolite standards (benzaldehyde, benzyl alcohol, and benzoic
acid) were obtained from Sigma Chemical Co. (St. Louis, MO).
Methylamine, 4-hydroxybenzaldehyde, and sodium cyanoboro-
hydride were purchased from Lancaster Chemical Co. (Windham,
NH), and sodium nitrite was purchased from Fluka Chemical
Co. (Ronkonkoma, NY). All other chemicals were purchased
from Sigma Chemical Co. or Aldrich Chemical Co.
crosomes (REM) metabolized NBzMA almost exclusively
by methylene hydroxylation (6). This is in contrast to
hepatic microsomal hydroxylation of NBzMA, which
occurs at both the methyl and methylene carbon (6, 11,
16). NBzMA metabolism by REM was inhibited more
than 95% by CO and 70% by SKF 525-A, classic P450
inhibitors (6). Therefore, rat esophageal NBzMA meth-
ylene hydroxylation is believed to be P450-mediated.
However, what P450 catalyzes this reaction is unknown.
Human P450 2E1, expressed in Hep G2 cells using
vaccinia virus, metabolizes NBzMA with a Km of 47 µM
(17). The products of the reaction were not characterized.
We report here, using baculovirus Sf9-expressed P450s,
that P450 2A6 catalyzes the metabolism of 1 µM NBzMA
at a rate more than 10 times greater than that of P450
2E1. P450 2A6 is a human liver P450 that is an efficient
and selective catalyst of the 5′-hydroxylation of another
esophageal carcinogen, NNN (18). The rat P450 that is
most homologous to P450 2A6 is the extrahepatic P450,
2A3 (19). P450 2A3 is a major P450 in rat nasal mucosa,
a tissue that efficiently metabolizes NBzMA (20, 21).
Related P450 2As in other species metabolize a number
of nitrosamines (22-25). Therefore, P450 2A3 appears
to be a likely candidate for the enzyme catalyzing
nitrosamine metabolism in the rat esophagus.
We recently reported that P450 2A3 mRNA is present
in the rat esophagus, albeit at very low levels (26). P450
2A6 is an efficient coumarin 7-hydroxylase (27), while
P450 2A3 was reported to be a less efficient catalyst of
this reaction (28). However, the kinetic parameters of
coumarin 7-hydroxylation by P450 2A3 have not been
determined. In the study reported here, we determined
that P450 2A3 and P450 2A6 are equally good catalysts
of both coumarin 7-hydroxylation and NBzMA methylene
hydroxylation. In contrast, REM efficiently catalyze the
methylene hydroxylation of NBzMA, but not the 7-hy-
droxylation of coumarin.
Syn th esis of N(o-OHBz)MA, N(m -OHBz)MA, a n d N(p-
OHBz)MA. The corresponding amines were synthesized by the
method of Borch et al. (29). Briefly, 4-hydroxybenzaldehyde (4
mmol) and methylamine hydrochloride (20 mmol) were dissolved
in 10 mL of absolute methanol, and the mixture was stirred at
room temperature for 1 h. Sodium cyanoborohydride (4 mmol)
was then added, and the reaction mixture was stirred overnight
under N2. The methanol was evaporated in a stream of N2, and
the resulting yellow oil was dissolved in 5 mL of 2 N HCl. This
mixture was extracted with ether (2 × 5 mL), and then the
aqueous phase was adjusted to pH >10 with 6 N KOH and
extracted again with ether (3 × 10 mL). The organic phase was
dried (MgSO4) and concentrated. The resulting yellow oil,
containing the amine, was dissolved in 2 mL of 30% acetic acid;
sodium nitrite (9.6 mmol) was added, and the solution was
stirred for 90 min at room temperature, as per Peterson et al.
(15). The reaction mixture was extracted with methylene
chloride (3 × 6 mL), washed with H2O (2 × 6 mL), dried
(MgSO4), and concentrated.
N(o-OHBz)MA and N(m-OHBz)MA were synthesized in the
same manner. The crude extracts from all three hydroxylated
nitrosamines were analyzed independently by LC/MS (system
IV, ammonium acetate buffer). The hydroxylated nitrosamine
peaks were identified by their characteristic mass spectra. N(o-
OHBz)MA [m/z (relative abundance)]: 199 ([M + Na]+, 14), 184
([M + NH4]+, 42), 167 ([M + H]+, 100), 121 ([C7H7NO]+, 6), 107
([C7H7O]+, 14). The same mass spectra were obtained for both
N(m-OHBz)MA and N(p-OHBz)MA with some differences in
relative abundance.
The identified hydroxynitrosamines were co-injected with the
products of P450 2A6-catalyzed NBzMA metabolism. Only N(p-
OHBz)MA coeluted with any metabolite peak, and therefore, it
was further purified by preparative TLC. Silica plates (Uniplate,
Exp er im en ta l P r oced u r es
Ca u tion : NBzMA is a carcinogen in experimental animals
and should be handled with care.

1256 Chem. Res. Toxicol., Vol. 12, No. 12, 1999
von Weymarn et al.
Analtech, Newark, DE) were used with a mobile phase of 1%
methanol and 99% CHCl3. The identity and purity of the product
were confirmed by HPLC using both systems I and II, LC/MS,
esophagi. The metabolism of NBzMA by these microsomes was
determined over a range of concentrations (0.5-1000 µM).
Microsomes (40-120 µg) were incubated with varying concen-
trations of [3H-benzyl]NBzMA as described above. The amounts
of the products of the reaction were determined to be linear with
time and protein concentration. Metabolites were analyzed by
reverse phase HPLC with radioflow detection using HPLC
systems I and II. The extent of inhibition of NBzMA activity by
carbon monoxide was determined. CO was bubbled gently into
a crimped vial containing REM in 100 mM sodium phosphate
buffer (pH 7.4) for 10 min prior to analysis of NBzMA metabo-
lism. The effect of coumarin on NBzMA metabolism was
measured. Coumarin, 10 or 100 µM, was either preincubated
for 15 min with REM and the NADPH generating system at
37 °C or added to the complete reaction mixture at time zero.
Since coumarin was dissolved in ethanol, control reaction
mixtures contained ethanol. The highest concentration of etha-
nol that was used was 0.75%. The coumarin 7-hydroxylase
activity of REM (2 mg/mL) with 40 µM coumarin was deter-
mined using the assay described above for P450 2A3. Immu-
noinhibition of REM-catalyzed NBzMA metabolism was carried
out using a polyclonal antibody to P450 2A5 [kindly provided
by M. Lang, Lyon, France (32)] and a monoclonal antibody to
P450 2E1 [1-91-3, kindly provided by H. Gelboin, National
Cancer Institute, Bethesda, MD (33)]. The concentration of each
antibody that inhibited P450 2A3- or P450 2E1-catalyzed
NBzMA metabolism by 70-80% was determined. That concen-
tration was then used in REM antibody inhibition experiments.
1
LC/MS/MS, and 1H NMR (600 MHz). H NMR (CD2Cl2) of the
E-isomer (80%): δ 2.89 (3H, s, CH3), 4.95 (1H, bs, OH), 5.19
1
(2H, s, CH2), 6.81 (2H, d, Ar-3,5 H), 7.12 (2H, d, Ar-2,6 H). H
NMR (CD2Cl2) of the Z-isomer (20%): δ 3.63 (3H, s, CH3), 4.69
(2H, s, CH2), 4.95 (1H, bs, OH), 6.77 (2H, d, Ar-3,5 H), 7.01 (2H,
d, Ar-2,6 H). MS [m/z (relative abundance)]: 184 ([M + NH4]+,
8), 167 ([M + H]+, 8), 121 ([C7H7NO]+, 100), 107 ([C7H7O]+, 64),
77 (C6H5+, 6). MS/MS of the ion at m/z 167 [m/z (relative
+
abundance)]: 121 ([C7H7NO]+, 16), 107 ([C7H7O]+, 100), (C6H5
18).
,
NBzMA Meta bolism by BV-Exp r essed P 450 2A6 a n d
P 450 2A3. P450 2A6 and 2A3 were expressed in the baculovirus
Sf9 insect system, and cell membranes were prepared as
previously described (18, 28). For all metabolism studies, BV-
expressed P450 2A6 or P450 2A3 (0.2-20 pmol) was preincu-
bated with BV-expressed human NADPH-P450 oxidoreductase
(3-300 pmol, Panvera Corp., Madison, WI) for 5 min at 37 °C.
A ratio of reductase to P450 that maximized the rate of product
formation was used for all analyses. Following preincubation,
the enzymes, NBzMA (1-80 µM), and an NADPH generating
system (0.4 mM NADP+, 100 mM glucose 6-phosphate, and 0.4
unit/mL glucose-6-phosphate dehydrogenase) in 100 mM sodium
phosphate buffer (pH 7.4) were incubated for 10 min at 37 °C.
The total volume was 0.2 mL. The reaction was terminated by
the addition of 20 µL of 0.3 M zinc sulfate and 20 µL of 0.3 M
barium hydroxide; the mixture was centrifuged for 10 min at
16000g. Semicarbazide (20 mM final concentration, unless
otherwise noted) and metabolite standards were added to the
supernatant, which was then frozen at -20 °C until analysis
by reverse phase HPLC with radioflow detection was carried
out. HPLC systems I and II were used and are described below.
The metabolism of [3H]benzyl alcohol (1 µM) was determined
using the same conditions as those described for NBzMA. [3H]-
Benzyl alcohol was generated by NBzMA metabolism with rat
liver microsomes and isolated by HPLC.
Kin etics a n d Sta tistica l An a lysis. Km and Vmax for NBzMA
R-hydroxylation and coumarin 7-hydroxylation were determined
using the EZ-fit 5 kinetics program from Perrella Scientific
(Amherst, NH) (34). This program uses a nonlinear regression
method of curve fitting and the Runs test of residuals to
determine statistically whether experimental data are randomly
distributed around the curve with 95% confidence.
HP LC System s. System I used a C18 Waters µBondapak
reverse phase column (3.9 mm × 300 mm, Waters Corporation,
Milford, MA) eluted with a linear gradient from 100% A [10 mM
ammonium acetate (pH 7)] to 50% B (methanol) over the course
of 40 min. Systems II-IV used a 10 µm Phenomenex-Bondclone
C18 column (3.9 mm × 300 mm). For system II, NBzMA
metabolites were eluted with a linear gradient from 95% A [1%
acetic acid (pH 2.7)] and 5% B (methanol) to 60% A and 40% B
over the course of 40 min. System III used isocratic elution with
92% 10 mM ammonium acetate (pH 7) and 8% methanol. N(4-
OHBz)MA eluted at 29 min on this system. System IV, for
7-hydroxycoumarin analysis, used an isocratic mobile phase
(30% methanol and 70% aqueous 0.2% acetic acid). The flow
rate was 1 mL/min for all systems.
NBzMA Meta bolism by BV-Exp r essed P 450 2E1 a n d
P 450 3A4. BV-expressed P450 2E1 membranes were prepared
previously (30), and P450 3A4 coexpressed with NADPH-P450
oxidoreductase was purchased from Gentest Corp. (Woburn,
MA). NBzMA (1 µM) metabolism was analyzed as described
above, except b5 at a 3:1 molar ratio of b5 to P450 was included
in the P450 2E1 analysis and no additional reductase was added
to the P450 3A4 analysis.
Cou m a r in 7-Hyd r oxyla tion by P 450 2A3. Coumarin (0.2-
500 µM) was incubated for 30 min at 37 °C with P450 2A3 (2-
10 pmol), BV-expressed human NADPH-P450 oxidoreductase
(10-60 pmol, preincubated as described above), and an NADPH
generating system in 0.2 mL of 100 mM sodium phosphate
buffer (pH 7.4). The reactions were stopped by the addition of
0.02 mL of 15% TCA. The reaction rate was maximal at a P450:
reductase ratio of 1:30. Reaction rates were determined to be
linear with P450 concentration and time. The 7-hydroxycou-
marin that was formed was analyzed by HPLC (system IV
described below) with fluorescence detection (excitation wave-
length of 350 nm and emission wavelength of 453 nm). Quan-
titation was by comparison to a standard curve of 7-hydroxy-
coumarin versus peak area. The extent of inhibition of P450
2A3-catalyzed coumarin 7-hydroxylation by NBzMA (5, 25, and
50 µM) was determined; the coumarin concentration was varied
from 0.5 to 100 µM.
Esop h a gu s Micr osom e P r ep a r a tion a n d NBzMA En -
zym e Assa ys. Esophagi from male Fischer 344 rats were
obtained from Harlan Bioproducts (Indianapolis, IN). Mi-
crosomes (0.5-3 mg of protein/mL) resuspended in 50 mM Tris-
HCl (pH 7.0) containing 1.15% KCl and 1 mM EDTA were
prepared as described previously (31). The protein concentration
was determined using Coomassie Plus Protein Assay Reagent
(Pierce, Rockford, IL), and the amount of P450 was determined
as described previously (6). REM were prepared from 5-15
LC/MS a n d LC/MS/MS An a lysis. MS analysis was carried
out on a Finnegan TSQ 7000 instrument with an APCI source
interfaced with an Alliance HPLC system (Waters Corp.). The
APCI settings were as follows: capillary temperature, 200 °C;
vaporizer temperature, 250 °C; and current, 5 µA. The instru-
ment was tuned to polypropylene glycol. A collision energy of
24 eV with a collision gas pressure of 1.06 mTorr was used for
MS/MS. LC conditions were as described for system I except a
2 mm × 300 mm Waters µBondapak C18 column was used with
a flow rate of 0.2 mL/min.
Resu lts
Met a b olism of NBzMA b y BV-E xp r essed P 450
2A6. In preliminary experiments using BV-expressed
enzymes, we determined that P450 2A6 was a better
catalyst of NBzMA metabolism than P450 2E1 or 3A4.
The rate of 1 µM NBzMA metabolism by P450 2A6 was
3.6 nmol min-1 nmol of P450-1 compared to rates of 0.2
and 0.12 nmol min-1 nmol of P450-1 for P450 2E1 and
3A4, respectively (Table 1). The products of [3H-benzyl]-
NBzMA metabolism were analyzed by reverse phase

NBzMA Hydroxylation by P450 2A3 and Rat Esophagus
Chem. Res. Toxicol., Vol. 12, No. 12, 1999 1257
Ta ble 1. P r od u cts of 1 µM NBzMA Meta bolism a
metabolitesd (percent of total)
enzymeb
ratec
3.5 min peak
benzyl alcohol
N(p-OHBz)MA
benzoic acid
benzaldehyde
P450 2E1
P450 3A4
P450 2A6
P450 2A3
REM
0.20
0.12
3.6
15
<0.5
3.0 ( 0.8
2.0 ( 0.5
32 ( 1.7
22
<0.5
9.0 ( 1.5
16 ( 0.8
4.0 ( 0.6
2
44
86
18
13
62 ( 2.9
66 ( 1.2
<1
<0.5
20 ( 3.2
3.0 ( 0.8
<1
3.0 ( 1.3
6.0 ( 0.5
65 ( 1.9
4.9
0.03e
a
BV-expressed P450s or REM were incubated for 10 min with 1 µM [3H-benzyl]NBzMA with no semicarbazide, and the metabolites
were analyzed by radioflow HPLC following the addition of 20 mM semicarbazide. Details are as described in Experimental Procedures.
P450 concentrations (picomoles per milliliter) were 100 (2E1), 25 (3A4), 2 (2A6), and 4 (2A3). c Nanomoles per minute per nanomole of
b
d
P450. Values are either the mean of duplicate determinations (2E1 and 3A4) or the average of three independent analyses analyzed in
duplicate (2A6, 2A3, and REM). e Nanomoles per minute per milligram of protein; the P450 concentration was not determined on this
REM preparation.
Ta ble 2. Kin etic P a r a m eter s of Cou m a r in
7-Hyd r oxyla tion a n d NBzMA Meth ylen e Hyd r oxyla tion a
Vmax (nmol min-1
substrate
enzyme
Km (µM)
nmol of P450-1
)
Vmax/Km
coumarinb P450 2A3c
P450 2A6d
1.7 ( 0.41
6.0 ( 1.2
1.7 ( 0.08
2.1 ( 0.1
1.0
0.4
7.6
2.3
NBzMA
P450 2A3c,e 0.63 ( 0.18
4.8 ( 0.2
P450 2A6f
6.7 ( 2.9
15.7 ( 1.9
a
The enzyme assay conditions and the quantitation of the
products of the reactions, 7-hydroxycoumarin and the sum of
benzaldehyde and benzoic acid, were as described in Experimental
Procedures. Kinetic and statistical analysis were carried out with
the Ez-Fit program. Values are means ( SE (Runs test p < 0.05).
b
The coumarin concentrations were 0.5, 1.0, 3.0, 5.0, 10, 20, 40,
and 100 µM. c Kinetic parameters are the mean of three indepen-
dent determinations. Values are from Zhou et al. (35). e The
d
F igu r e 1. Radioflow HPLC analysis of 2 µM NBzMA metabo-
lism by BV-expressed P450 2A6. HPLC system I (pH 7.0) was
used. Arrows denote the retention times of co-injected standards.
Semicarbazide was added prior to HPLC analysis; therefore, the
peak labeled benzaldehyde is the benzaldehyde semicarbazone.
concentrations of NBzMA were 0.25, 0.5, 1.0, 2.0, 3.0, 5.0, 8.0, 10,
20, 40, and 80 µM. f The concentrations of NBzMA were 1.0, 3.0,
5.0, 10, 20, 40, and 80 µM; the values are means of two
independent determinations.
synthesized the three isomers of ring-hydroxylated
NBzMA. The metabolite eluting at 25 min in Figure 1
coeluted with N(p-OHBz)MA (Scheme 1). The ortho and
meta isomers [N(o-OHBz)MA and N(m-OHBz)MA] eluted
at 29 and 30 min, respectively. The P450 2A6 metabolite
eluting at 25 min (10-15 ng) was collected from HPLC
and analyzed by LC/MS and LC/MS/MS. The spectra that
were obtained were identical to those of the synthesized
N(p-OHBz)MA standard.
HPLC with radioflow detection. Semicarbazide was not
routinely included in the enzyme reaction since it had
little or no effect on product distribution when short
incubation times were used. It was added immediately
after terminating the reaction to trap the benzaldehyde.
Four metabolite peaks were detected as products of
P450 2A6-catalyzed NBzMA metabolism (Figure 1). The
major radioactive peak, eluting at 32 min, coeluted with
benzaldehyde semicarbazone. If semicarbazide was not
added to the sample prior to HPLC analysis, the radioac-
tive peak at 32 min eluted at 29.5 min, the retention time
of benzaldehyde. Two of the remaining products also
coeluted with known NBzMA metabolites, benzoic acid
(tR of 7 min, Figure 1) and benzyl alcohol (tR of 18 min,
Figure 1), in two different HPLC systems, I and II. The
rate of benzyl alcohol formation was not affected by the
inclusion of semicarbazide in the incubation mixture.
Also, benzyl alcohol was not further metabolized to either
benzaldehyde or benzoic acid. Therefore, benzyl alcohol
is the product of NBzMA methyl hydroxylation and not
the product of benzaldehyde reduction, and benzaldehyde
and benzoic acid are products of methylene hydroxyla-
tion.
N(p-OHBz)MA has not previously been reported to be
a metabolite of NBzMA. However, it was the second most
abundant product of P450 2A6-catalyzed NBzMA me-
tabolism, accounting for 20% of the total metabolites that
were detected (Table 1). The products of methylene
hydroxylation, benzaldehyde and benzoic acid, accounted
for 65% of the products, while benzyl alcohol, the product
of methyl hydroxylation, accounted for 9%. The kinetic
parameters for P450 2A6-catalyzed NBzMA R-hydroxy-
lation were determined. The ratio of methyl to methylene
hydroxylation products was constant over the range of
concentrations that was used. The Km for methylene
hydroxylation was 6.7 µM and the Vmax 15.7 nmol min-1
nmol of P450-1 (Table 2). The kinetic parameters for
coumarin 7-hydroxylation were not determined on the
same enzyme preparation. However, using this prepara-
tion and the same conditions that were used to study
NBzMA metabolism, the rate of 7-hydroxylation of 40 µM
coumarin was 3.5 nmol min-1 nmol of P450-1. This
coumarin concentration is well above the reported Km of
6.7 µM (35). Therefore, P450 2A6 appears to be a better
catalyst of NBzMA methylene hydroxylation than of
coumarin 7-hydroxylation.
The peak at 25 min did not elute with any previously
identified NBzMA metabolite. We hypothesized that this
peak might be a product of ring hydroxylation of NBzMA.
This metabolite was collected and analyzed by LC/MS
with APCI positive ion detection. The expected proto-
nated molecular ion ([M + H]+, m/z 167) was detected,
as well as a major fragment ion (m/z 107, [C7H7O]+).
These data are consistent with the identity of this
metabolite being hydroxylated NBzMA. Therefore, we

1258 Chem. Res. Toxicol., Vol. 12, No. 12, 1999
von Weymarn et al.
F igu r e 2. Competitive inhibition of P450 2A3-catalyzed cou-
marin 7-hydroxylation by NBzMA. The formation of 7-hydroxy-
coumarin was assessed by HPLC analysis with fluorescence
detection. NBzMA was added at the indicated concentrations.
Curves were graphed by nonlinear regression kinetic param-
eters determined using Ez-Fit (34). All values are the averages
of duplicate determinations.
Meta bolism of Cou m a r in a n d NBzMA by BV-
Exp r essed P 450 2A3. P450 2A3 is the rat P450 that is
most homologous to P450 2A6 (19, 27, 36). As noted
above, P450 2A6 is an efficient and selective coumarin
hydroxylase (27). We therefore characterized the cou-
marin 7-hydroxylase activity of P450 2A3. The level of
formation of 7-hydroxycoumarin was measured using
reverse phase HPLC with fluorescence detection. P450
2A3 efficiently catalyzed coumarin 7-hydroxylation [Km
) 1.7 µM and Vmax ) 1.7 nmol min-1 nmol of P450-1
(Table 2)]. The catalytic efficiency is 1.0, which is similar
to that reported previously for BV-expressed P450 2A6
[Table 2 (35)]. The effect of NBzMA on P450 2A3-
catalyzed coumarin 7-hydroxylation was determined, and
significant inhibition was observed with 5 µM NBzMA.
The type of inhibition was competitive, and the Ki was
0.21 µM (Figure 2).
F igu r e 3. Radioflow HPLC analysis of the products of (A) 2
µM NBzMA metabolism by BV-expressed P450 2A3 and (B)
REM-catalyzed 1 µM NBzMA metabolism. HPLC system II (pH
2.9) was used. Arrows denote the retention time of co-injected
standards. Semicarbazide was added prior to HPLC analysis;
therefore, the peak labeled benzaldehyde is the benzaldehyde
semicarbazone.
Ta ble 3. Kin etic P a r a m eter s of NBzMA Meth ylen e
Hyd r oxyla tion by REMa
The metabolism of [3H-benzyl]NBzMA by P450 2A3
was also characterized. The products of this reaction were
analyzed by reverse phase HPLC and shown to coelute
with standards using either system I (pH 7.0) or II (pH
2.7). The chromatogram illustrated in Figure 3A was
obtained using HPLC system II. Like P450 2A6, the
major product of P450 2A3-catalyzed NBzMA metabolism
was benzaldehyde (29 min, Figure 3A). However, in
contrast to P450 2A6, significantly more benzyl alcohol
(12 min) and much less N(p-OHBz)MA (tR ) 16 min) were
generated by P450 2A3-catalyzed NBzMA metabolism
(Table 1). In addition, an early eluting peak (tR ) 4.5 min)
was detected. The size of this peak was variable, and a
portion was often present in the control. This peak, which
eluted 1 min past the void volume, was not further
characterized.
As was true for P450 2A6, the rate of benzyl alcohol
formation was unaffected by the addition of semicarbi-
zide. Benzyl alcohol was also not further metabolized by
P450 2A3. The ratio of benzaldehyde to benzyl alcohol
was 4:1 over the range of concentrations that was used
to determine the kinetic parameters for P450 2A3-
catalyzed NBzMA methylene hydroxylation. The Km (0.63
µM) for P450 2A3 was 10-fold lower than that of P450
2A6, and the Vmax (4.8 nmol min-1 nmol-1) was 3-fold
lower (Table 2). Therefore, P450 2A3 had a catalytic
efficiency for the methylene hydroxylation of NBzMA that
is 3-fold greater than that of P450 2A6.
REM
[NBzMA]
(µM)
preparationb
Km (µM)
Vmax (nmol min-1 mg-1
)
1
2
2
0.5-40
0.5-40
0.5-1000 446 ( 143
3.8 ( 0.42
5.1 ( 0.94
0.38 ( 0.13
0.054 ( 0.003
0.50 ( 0.07
a
The enzyme assay conditions and the quantitation of the
products of the reactions, 7-hydroxycoumarin and the sum of
benzaldehyde and benzoic acid, were as described in Experimental
Procedures. Kinetic and statistical analysis were carried out with
the Ez-Fit program. Values are means ( SE (Runs test p < 0.05).
b
Two preparations (1 and 2) of REM were analyzed with 0.5, 1.0,
3.0, 5.0, 10, 20, and 40 µM NBzMA, and preparation 2 was also
analyzed with additional concentrations of 80, 150, 500, and 1000
µM. Rates were the mean of two determinations.
3B) and an early eluting peak (tR ) 3.5 min). No
benzaldehyde was detected whether semicarbazide was
included in the incubation or not. We also observed that
semicarbazide (1-20 mM) inhibited REM-catalyzed
NBzMA metabolism; therefore, it was not included in the
enzyme reaction mixtures. A small amount of benzyl
alcohol was detected (Table 1 and Figure 3B).
The kinetic parameters of NBzMA methylene hydroxy-
lation were determined for two different REM prepara-
tions. The protein concentration of these two preparations
was about 4-fold different. Using REM preparation 1 and
NBzMA concentrations of 0.5-40 µM, the rate of product
formation appeared to be maximized. The Km was 3.8 (
0.42 µM and Vmax 0.38 ( 0.13 nmol min-1 mg-1 (Table
3). The same analysis was repeated on a new REM
preparation (2), and for NBzMA concentrations of 0.5-
40 µM, Km was determined to be 5.1 ( 0.94 µM and Vmax
0.054 ( 0.003 nmol min-1 mg-1. The difference in Vmax
is most likely due to the different protein concentrations
Meta bolism of NBzMA by Ra t Esop h a gea l Mi-
cr osom es. REM catalyzed the metabolism of NBzMA,
and this metabolism was completely inhibited by carbon
monoxide (Table 4). Two major metabolites were detected
by radioflow HPLC, benzoic acid (tR ) 19 min, Figure

NBzMA Hydroxylation by P450 2A3 and Rat Esophagus
Chem. Res. Toxicol., Vol. 12, No. 12, 1999 1259
Ta ble 4. In h ibition of 1 µM NBzMA Meta bolism by Ra t
metabolism (38), and P450 2E1 does metabolize NBzMA
(Table 1). However, antibody to P450 2E1 did not inhibit
REM-catalyzed NBzMA metabolism. Also, the distribu-
tion of NBzMA metabolites formed by P450 2E1 was not
the same as that of metabolites formed by REM. P450
3A4, like the esophagus, metabolized NBzMA predomi-
nantly to benzoic acid (Table 1), and this metabolism was
completely inhibited by 20 mM semicarbazide (data not
shown).
Esop h a gea l Micr osom esa
inhibitor
percent inhibitionb
100
carbon monoxide
10 µM coumarin
without preincubation
with preincubationc
100 µM coumarind
without preincubation
with preincubationc
0.75% ethanold
11
63
72
83
without preincubation
with preincubationc
P450 2A5 antibodye
P450 2E1 antibodye
20 mM semicarbazide
23
22
0
0
40
Discu ssion
We report here that the human coumarin 7-hydroxy-
lase, P450 2A6, and the closely related rat enzyme P450,
2A3, are efficient catalysts of NBzMA metabolism. The
major metabolic pathway catalyzed by both enzymes is
methylene hydroxylation (Table 1), the proposed activa-
tion pathway for this carcinogenic nitrosamine (5, 12).
In addition, we characterized a previously unidentified
metabolite of NBzMA, the ring hydroxylation product,
N(p-OHBz)MA (Scheme 1). Para hydroxylation of the
benzyl ring was a major pathway of P450 2A6-catalyzed
NBzMA metabolism, occurring at a rate that was about
1/3 of that of methylene hydroxylation. In contrast, P450
a
Reaction conditions were as described in Experimental Pro-
cedures. Values are reported as the average of duplicate deter-
b
minations. Total metabolism was assessed; all products were
affected to the same extent. c All reagents except NBzMA were
incubated for 15 min prior to the initiation of the reaction with
d
NBzMA. Coumarin was dissolved in 0.75% ethanol. e The con-
centration of antibodies was sufficient to inhibit P450 2A3 or P450
2E1 NBzMA metabolism >70%.
of the preparations which do not necessarily parallel the
P450 content or activity. We have previously observed
variable rates, expressed per milligram of protein, with
REM and NNN metabolism (7). To compare our results
to those of Labuc and Archer, who determined the rates
of REM-catalyzed NBzMA metabolism at millimolar
concentrations, we determined the rate of NBzMA me-
tabolism up to concentrations of 1000 µM. The products
of the reaction were generated in the same ratio at all
concentrations, and the rate of methylene hydroxylation
at 1000 µM increased 8-fold from that at 40 µM. Kinetic
parameters were determined over this concentration
range; the Km was 450 ( 143 µM and Vmax 0.5 ( 0.07
nmol min-1 mg-1 (Table 3).
The possible role of P450 2A3 in the metabolism of
NBzMA by REM was investigated by a series of inhibi-
tion experiments. Coumarin (10 or 100 µM) inhibited
NBzMA metabolism by 11 or 72%, respectively (Table 4).
The extent of inhibition by 10 µM coumarin increased
from 11 to 63% when REM were preincubated with
coumarin for 15 min. Coumarin (40 µM) was not me-
tabolized to 7-hydroxycoumarin by the esophagus. The
limit of detection for this analysis was 0.13 pmol of
7-hydroxycoumarin min-1 mg of REM-1. The same REM
preparation catalyzed the methylene hydroxylation of 2
µM NBzMA at a rate of 300 pmol min-1 mg-1. Therefore,
if REM catalyze the 7-hydroxylation of coumarin at all,
it is at a rate that is more than 1000-fold lower than the
rate of NBzMA methylene hydroxylation. This is in
contrast to the relative catalytic activities of P450 2A3
for these two reactions (Tables 2). These data do not
support a role for P450 2A3 in the REM-catalyzed
metabolism of NBzMA. Further support for this conclu-
sion is that an antibody to P450 2A5 did not inhibit
NBzMA metabolism by REM under conditions that
inhibited P450 2A3-catalyzed metabolism more than 80%
(Table 4). P450 2A5 is a mouse coumarin 7-hydroxylase,
which is more that 95% homologous with P450 2A3 (19,
36, 37).
2A3 catalyzed the formation of N(p-OHBz)MA at a rate
1
that was less than
hydroxylation.
/
of that of NBzMA methylene
20
Previously, BV-expressed P450 2A3 was reported to
have a low rate of coumarin 7-hydroxylation activity
relative to that of BV-expressed P450 2A6 (28). However,
we report here that P450 2A3 is an efficient catalyst of
coumarin 7-hydroxylation. In the previous study, the rate
of coumarin 7-hydroxylation by P450 2A3, reconstituted
with rabbit NADPH-P450 oxidoreductase, was deter-
mined at a single coumarin concentration of 100 µM. We
have found in more recent studies that the rate of P450
2A3 catalyzing coumarin 7-hydroxylation with rat re-
ductase is at least 3 times greater than that with rabbit
P450 reductase.2 Furthermore, in the previous study, the
optimal ratio of P450 to reductase was determined with
testosterone as a substrate, and the highest ratio that
was used was 6:1 (28); further increasing this ratio, as
in the study presented here (30:1), may have been
necessary to obtain maximal activity for coumarin 7-hy-
droxylation. In addition, the method of 7-hydroxycou-
marin analysis used in this study was HPLC, which may
have allowed more accurate quantitation.
REM were efficient catalysts of NBzMA methylene
hydroxylation (Table 3). The kinetic data suggest that
more than one enzyme may be catalyzing this reaction.
The Km calculated at low NBzMA concentrations was 10-
20-fold lower than those reported previously for two other
rat esophageal carcinogens, NNN and NMAA (7, 9).
However, if the concentration of NBzMA was increased
above 40 µM, the rate of methylene hydroxylation
increased, despite the fact that the rate appeared to
saturate at lower concentrations. These data suggest the
presence of more than one enzyme in REM that catalyzes
the methylene hydroxylation of NBzMA. We had hypoth-
esized that P450 2A3 may be one of these enzymes.
However, P450 2A3 is an efficient catalyst of coumarin
7-hydroxylation, while REM did not catalyze this reaction
at all. In addition, REM-catalyzed NBzMA metabolism
was not inhibited by the P450 2A5 antibody. Therefore,
The REM-catalyzed metabolism of NBzMA was inhib-
ited more than 40% by 20 mM semicarbazide (Table 4).
However, the same amount of semicarbazide had no
effect on NBzMA metabolism by either P450 2A3 or 2A6
(data not shown). Semicarbazide inhibits P450 2E1
2
X. Zhuo and X. Ding, unpublished results.

1260 Chem. Res. Toxicol., Vol. 12, No. 12, 1999
von Weymarn et al.
P450 2A3 is not catalyzing the metabolism of NBzMA in
the rat esophagus.
NBzMA metabolism. In contrast, these experiments do
provide some evidence that support investigating a
possible role for a P450 related to P450 3A4 in REM
NBzMA metabolism. However, while P450 3A5 has been
detected in human esophagus (42), P450 3A enzymes
have not been detected in the rat esophagus (43).
Surprisingly, coumarin did inhibit REM-catalyzed
NBzMA metabolism, and interestingly, the extent of this
inhibition increased significantly when REM were pre-
incubated with coumarin. These data suggest that a
metabolite of coumarin was responsible for the inhibition
that was observed. Rat hepatic microsomes, which do not
contain P450 2A3, primarily metabolize coumarin to
o-hydroxyphenylacetaldehyde (39). The 3,4-epoxide of
coumarin is considered the key intermediate in the
formation of o-hydroxyphenylacetaldehyde (40, 41). There-
fore, it may be that the coumarin inhibition of REM-
catalyzed NBzMA metabolism we observed is due to the
generation of the reactive 3,4-epoxide of coumarin (41).
The same enzyme that catalyzes the epoxidation of
coumarin may also catalyze NBzMA metabolism in the
esophagus. We plan to investigate this hypothesis in the
future. Several P450s, including 1A1, 2E1, and 3A4,
catalyze this reaction (35).
Another distinguishing feature of REM-catalyzed
NBzMA metabolism was the generation of benzoic acid
and not benzaldehyde. These data suggest that the P450
generating benzaldehyde is an equally good catalyst of
benzaldehyde oxidation. We were unable to trap benzal-
dehyde even with 20 mM semicarbazide. This is in
contrast to what was reported by Labuc and Archer (6),
who quantified benzaldehyde as the major product of
REM-catalyzed NBzMA metabolism. However, they were
assessing NBzMA metabolism at a much higher concen-
tration (5 mM) than we used in our studies. Consistent
with the kinetic parameters of NBzMA methylene hy-
droxylation reported here, one P450 may be the major
catalyst of this reaction at a NBzMA concentration of 40
µM and a second at a NBzMA concentrations of 5 mM.
The existence of more than one rat esophageal P450 that
catalyzes NBzMA metabolism is also supported by the
fact that Labuc and Archer observed no effect of semi-
carbazide on NBzMA metabolism, while we observed
significant inhibition (Table 4).
A third unique feature of REM-catalyzed NBzMA
metabolism was the formation of a second major metabo-
lite (3.5 min peak, Table 1), which eluted in the void
volume. Although we have not confirmed the identity of
this metabolite, it is most likely tritiated water, with
which it coelutes (data not shown). The tritium is in the
meta position of the benzene ring of NBzMA, and
hydroxylation at this site would result in the release of
tritiated water. Therefore, benzyl ring hydroxylation at
the meta position may be a significant pathway of
NBzMA metabolism in the esophagus. This is in contrast
to the hydroxylation at the para position catalyzed by
P450 2A6. The relative extent and importance of benzyl
ring hydroxylation in REM-catalyzed NBzMA metabo-
lism are being investigated.
In summary, both REM and P450 2A3 are efficient
catalysts of NBzMA methylene hydroxylation. However,
several characteristics of REM-catalyzed NBzMA me-
tabolism provide strong evidence that P450 2A3 does not
contribute to this metabolism. (1) REM do not catalyze
the 7-hydroxylation of coumarin. (2) The antibody to P450
2A5 inhibits 2A3- but not REM-catalyzed NBzMA me-
tabolism. (3) The major product of NBzMA metabolism
by REM is benzoic acid and not benzaldehyde. Results
from preliminary experiments (Tables 1 and 3) also do
not support a role for P450 2E1 in REM-catalyzed
Ack n ow led gm en t. We thank Edward McIntee and
Lisa Peterson for useful discussions about N(p-OHBz)-
MA synthesis and Xiaoliang Zhuo for preparation of BV-
expressed P450 2A3. This research was supported by
NIH Grants CA74913 (S.E.M.) and ES07462 (X.D.). All
MS analyses were carried out in the Analytical Chem-
istry Core Facility of the University of Minnesota Cancer
Center (1P30-CA77598).
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