Mechanistic studies on the cytochrome P450-catalyzed dehydrogenation of 3- methylindole

Article abstract of DOI:10.1021/tx9501187

The mechanism of 3-methyleneindolenine (3MEI) formation from 3- methylindole (3MI) in goat lung microsomes was examined using stable isotope techniques. 3MEI is highly electrophilic, and its production is a principal factor in the systemic pneumotoxicity of 3MI. Noncompetitive intermolecular isotope effects of (D)V = 3.3 and (D)(V/K) = 1.1 obtained after deuterium substitution at the 3-methyl position indicated either that hydrogen abstraction from the methyl group was not the initial rate-limiting step or that this step was rate-limiting and was masked by a high forward commitment and low reverse commitment to catalysis. An intramolecular isotope effect of 5.5 demonstrated that hydrogen atom abstraction was probably the initial oxidative and rate-limiting step of 3MI bioactivation or that deprotonation of an aminium cation radical, produced by one-electron oxidation of the indole nitrogen, was rate-limiting. However, a mechanism which requires deprotonation of the aminium cation radical is probably precluded by an unusual requirement for specific base catalysis at a site in the cytochrome P450 enzyme other than the heme iron. The pattern of ¹⁸O incorporation into indole-3-carbinol from ¹⁸O₂ and H₂¹⁸O indicated that approximately 80% of the indole-3-carbinol was formed in goat lung microsomes by hydration of 3MEI. However, the inverse reaction, dehydration of indole-3-carbinol, did not significantly contribute to the formation of 3MEI. These results show that 3MEI was formed in a cytochrome P450-catalyzed dehydrogenation reaction in which the rate-limiting step was presumably hydrogen atom abstraction from the 3-methyl position. The ratio of the amounts of 3MEI to indole-3-carbinol formed (50:1) indicated that dehydrogenation of 3MI is an unusually facile process when compared to the dehydrogenation of other substrates catalyzed by cytochrome P450 enzymes.

Full text of DOI:10.1021/tx9501187

Chem. Res. Toxicol. 1996, 9, 291-297
291
Mech a n istic Stu d ies on th e Cytoch r om e P 450-Ca ta lyzed
Deh yd r ogen a tion of 3-Meth ylin d ole
†
Gary L. Skiles and Garold S. Yost*
Department of Pharmacology and Toxicology, 112 Skaggs Hall, University of Utah,
Salt Lake City, Utah 84112
Received J une 30, 1995X
The mechanism of 3-methyleneindolenine (3MEI) formation from 3-methylindole (3MI) in
goat lung microsomes was examined using stable isotope techniques. 3MEI is highly
electrophilic, and its production is a principal factor in the systemic pneumotoxicity of 3MI.
D
D
Noncompetitive intermolecular isotope effects of V ) 3.3 and (V/K) ) 1.1 obtained after
deuterium substitution at the 3-methyl position indicated either that hydrogen abstraction
from the methyl group was not the initial rate-limiting step or that this step was rate-limiting
and was masked by a high forward commitment and low reverse commitment to catalysis.
An intramolecular isotope effect of 5.5 demonstrated that hydrogen atom abstraction was
probably the initial oxidative and rate-limiting step of 3MI bioactivation or that deprotonation
of an aminium cation radical, produced by one-electron oxidation of the indole nitrogen, was
rate-limiting. However, a mechanism which requires deprotonation of the aminium cation
radical is probably precluded by an unusual requirement for specific base catalysis at a site in
1
8
the cytochrome P450 enzyme other than the heme iron. The pattern of O incorporation into
indole-3-carbinol from 18
O and H O indicated that approximately 80% of the indole-3-carbinol
18
2 2
was formed in goat lung microsomes by hydration of 3MEI. However, the inverse reaction,
dehydration of indole-3-carbinol, did not significantly contribute to the formation of 3MEI.
These results show that 3MEI was formed in a cytochrome P450-catalyzed dehydrogenation
reaction in which the rate-limiting step was presumably hydrogen atom abstraction from the
3
-methyl position. The ratio of the amounts of 3MEI to indole-3-carbinol formed (50:1) indicated
that dehydrogenation of 3MI is an unusually facile process when compared to the dehydro-
genation of other substrates catalyzed by cytochrome P450 enzymes.
1
The pulmonary toxicity of 3-methylindole (3MI) is at
least in part due to alkylation of cellular proteins by a
dehydrogenation product of its cytochrome P450-cata-
lyzed metabolism: 3-methyleneindolenine (4, Scheme 1;
Sch em e 1. P ossible Mech a n ism s of
3-Meth ylen ein d olen in e (4) F or m a tion fr om
-Meth ylin d ole (1) in Goa t Lu n g Micr osom es
a
3
3
MEI). The structure of 3MEI was first inferred when
a glutathione adduct was isolated from goat lung mi-
crosomal incubations of 3MI that were supplemented
with glutathione (1). Studies showed that covalent
binding of 3MI in goat lung microsomes was inversely
proportional to the concentration of supplemented glu-
tathione or cysteine in the reactions (2) and that the in
vivo toxicity of 3MI was proportional to depletion of
glutathione in goat (3) and rat lungs (4). Bioactivation
of 3MI to 3MEI was also shown to occur in vivo when a
mercapturic acid adduct that was produced subsequent
to glutathione conjugation of 3MEI was excreted from
animals treated with 3MI (5). 3MEI also reacts with
protein thiols, as shown by the isolation of a cysteine
adduct of 3MI from hydrolysates of goat and human
microsomal proteins that were covalently bound to 3MI
in a P450-catalyzed reaction (6). The formation of 3MEI
by cytochrome P450 enzymes has also been demonstrated
in incubations of 3MI in lysates of cell lines containing
a
Possible pathways are as follows: A, hydrogen atom abstrac-
tion, hydroxyl rebound, and dehydration of indole-3-carbinol (3);
B, hydrogen atom abstraction followed by a second one-electron
oxidation and proton loss; C, one-electron oxidation of the nitrogen
followed by loss of a 3-methyl proton and a one-electron oxidation;
and D, hydrogen atom abstraction and rearrangement of the
radical followed by hydroxyl rebound and dehydration of the
hemiaminal (6).
expressed enzymes from cloned human, rabbit, and
mouse P450s (7). The cytochrome P450-mediated pro-
duction of 3MEI was also shown in isolated rabbit lung
cells (8), where addition of 1-aminobenzotriazole, a mech-
anism-based inhibitor of cytochrome P450 enzymes,
essentially abolished the production of the glutathione
adduct of 3MEI. Thus, the formation of 3MEI appears
to be mediated by several cytochrome P450 enzymes from
*
Author to whom correspondence should be addressed.
Present address: Biochemical and Investigative Toxicology, De-
†
partment of Safety Assessment, WP45-319, Merck Research Labora-
tories, West Point, PA 19486.
X
Abstract published in Advance ACS Abstracts, December 15, 1995.
Abbreviations: 3-methylindole, 3MI; 3-methyleneindolenine, 3MEI;
1
indole-3-carbinol, I3COH; 3-[(N-acetyl-L-cystein-S-yl)methyl]indole,
MINAC; 3-([ H
; N-acetyl-L-cysteine, NAC; cytochromes P450, P450; N-(trimethyl-
silyl)trifluoroacetamide, MSTFA.
2
2
3
d
3 3 2
]methyl)indole, 3MI-d ; 3-([ H ]methyl)indole, 3MI-
2
0
893-228x/96/2709-0291$12.00/0 © 1996 American Chemical Society
+
+

2
92 Chem. Res. Toxicol., Vol. 9, No. 1, 1996
Skiles and Yost
several species. These and other mechanistic and toxi-
cological aspects of 3MI metabolism have been previously
reviewed (9, 10).
Exp er im en ta l Section
Ch em ica ls. 3-Methylindole, indole-3-carboxylic acid, indole-
-carboxaldehyde, N-acetyl-L-cysteine (NAC), acetanilide, p-
hydroxyacetanilide, benzoyl chloride (reagent grade), and H
95 atom %) were purchased from Aldrich Chemical Co. (Mil-
3
The mechanism by which 3MEI is formed from 3MI is
18
2
O
-
+
unclear. The P450-catalyzed reaction, 3MI + 2e + 2H
f 3MEI + 2H O, describes a net dehydrogenation,
(
+
O
2
2
waukee, WI). Indole-3-carbinol was purchased from Sigma
Chemical Co. (St. Louis, MO), and NADPH was purchased from
United States Biochemical (Cleveland, OH). N-Methyl-N-
(trimethylsilyl)trifluoroacetamide (MSTFA) and dry, silation-
grade acetonitrile were purchased from Pierce Chemical Co.
but 3MEI could be formed from indole-3-carbinol (3,
I3COH) that undergoes dehydration to form 3MEI
(Scheme 1, pathway A). Acid-catalyzed dehydration of
I3COH is a facile process and has been the subject of
extensive study because I3COH is present in cruciferous
vegetables and is suspected to act as an anticarcinogen
(
Rockford, IL). 18
2
O (50 atom %) was obtained from Cambridge
Isotope Laboratories (Woburn, MA). 3-[(N-Acetyl-L-cystein-S-
yl)methyl]indole (3MINAC) was synthesized as previously
(ref 11 and references therein). I3COH is a product
2
2
described (5), and 3-([ H
methyl)indole (3MI-d ) were synthesized by reduction of indole-
-carboxylic acid and indole-3-carboxyaldehyde, respectively,
3 3 2
]methyl)indole (3MI-d ) and 3-([ H ]-
formed during the P450-catalyzed metabolism of 3MI in
vitro (8), and indole-3-carboxylic acid, presumably derived
from I3COH, is a major product of the in vivo metabolism
of 3MI (12). The most simple and reasonable mechanism
of I3COH formation is via hydrogen radical abstraction
and hydroxyl rebound. The intermediary carbon-cen-
tered radical (2) that occurs during the formation of
I3COH might also undergo a one-electron oxidation to
directly form 3MEI (Scheme 1, pathway B) if enzymatic
and chemical conditions favor such a reaction. Alterna-
tively, I3COH may be formed by hydration of 3MEI.
A completely different process of 3MEI formation,
wherein an electron is abstracted from the nitrogen in
the initial step followed by loss of a proton and a one-
electron oxidation, is also possible (Scheme 1, pathway
C). Such a mechanism is reasonable because 3MI has
been shown to have a one-electron reduction potential of
2
3
4
with LiAlD according to a published method (16). Percent
isotopic incorporation was determined by gas chromatography/
mass spectrometry (vide infra) with the following results: 3MI-
d
d
2
0.0079 ((0.0516)% d0, 1.14 ((0.0501)% d
, 0.366 ((0.0351)% d ; 3MI-d 0.0613 ((0.0089)% d
, 97.9 ((0.0626)% d All other
1
, 98.5 ((0.0611)%
2
3
3
0
, d not
1
detected, 2.10 ((0.0604)% d
2
3
.
chemicals and reagents except those described below were
purchased from local suppliers in the highest purity available.
Micr osom e P r ep a r a tion . Goat lung and mouse liver mi-
crosomes (used for positive control experiments in the
18
2
O -
incorporation studies; described below) were prepared by stan-
dard centrifugal isolation procedures (17). Goat lungs were
obtained from mature female goats. Goats were sedated with
pentobarbital and killed by exsanguination. The lungs were
perfused in situ with ice-cold 0.05 M phosphate buffer (pH 7.4)
that contained 1 mM EDTA and 1.15% KCl, and then frozen at
-
80 °C. Mouse livers were obtained from male Swiss-Webster
1
.05V (vs H) in studies where the nitrogen-centered
mice (SASCO, Omaha, NE) that were killed by cervical disloca-
tion. The livers were rapidly removed and then minced in ice-
cold 0.05 M Tris (Sigma) buffer (pH 7.4, determined at 25 °C)
that contained 1.0 mM EDTA, 0.25 M sucrose, and 0.15 M KCl
cation radical was presumed to be the product of the one-
electron oxidation of 3MI by perchlorate radical (13).
When cytochromes P450-catalyzed monooxygenations
of xenobiotics occur at carbon atoms adjacent to, or
conjugated with, nitrogen atoms, several lines of evidence
have shown that the reactions proceed via an initial one-
electron oxidation of the nitrogen followed by loss of the
R or γ protons which are rendered more acidic by the
aminium radical (14, 15). After rearrangement of the
radical to the R or γ carbon, hydroxyl rebound can occur
to give a rearranged monooxygenated product. In lieu
of hydroxyl rebound, abstraction of a second electron to
yield a dehydrogenation product could also occur, par-
ticularly when the hydrogen is lost from the γ-position.
The monooxygenated and dehydrogenated products could
also be formed via an initial abstraction of the R or γ
hydrogens; however, low kinetic deuterium isotope effects
typically observed in these reactions have been inter-
preted to mean that nitrogen oxidation and not hydrogen
abstraction is the initial step in the reaction.
(
buffer 1). Goat lung tissue was blended in a Waring blender
in buffer 1 in a 4:1 buffer volume/tissue weight ratio. Blended
goat lung tissue and minced mouse livers were homogenized
with a Potter-Elvehjem homogenizer and then centrifuged at
000g for 30 min to remove cellular debris. The supernatant
was filtered through cheesecloth and then centrifuged at
05000g for 90 min. The microsomal pellets were resuspended
in buffer 2 (identical to buffer 1 except that it did not contain
KCl) in a 1:3 buffer volume/tissue weight ratio and then
centrifuged a second time at 105000g, before they were again
suspended in buffer 2 (1:3 dilution). The microsomal protein
content was determined with a BSA Protein Assay Reagent Kit
(Pierce), and the P450 content of the goat lung microsomes was
determined from the dithionite-reduced difference spectra of
carbon monoxide saturated microsomes as previously described
9
1
(
18). Microsomes were stored at -80 °C.
Deu ter iu m Isotop e Effect In cu ba tion s. Noncompetitive
intermolecular deuterium-isotope effects were determined by
incubating 0.05, 0.02, and 0.008 mM 3MI or 3MI-d
in goat lung
3
The studies reported herein probed the different pos-
sible mechanisms for the formation of 3MEI and I3COH
by determining the kinetic deuterium isotope effects in
microsomes (1.1 nmol of P450/mL), 1.0 mM NADPH, and 8.0
mM NAC in 0.05 M potassium phosphate buffer (pH 7.4) in a
total volume of 3.0 mL at 37 °C. 3MEI was trapped with NAC
to form 3MINAC because 3MEI is too reactive to quantify
directly. Substrate was introduced in 10-µL volumes of ethanol,
and incubations of each concentration of substrate were per-
formed in triplicate. The reactions were stopped after 2.0 min
by adding 3.0 mL of ice-cold acetonitrile. Denatured protein
was precipitated by centrifugation, and then the supernatant
was decanted and evaporated to about 1 mL in vacuo. The
residue was brought to a volume of 1.5 mL with the starting
HPLC mobile phase and analyzed directly (vide infra).
3
MEI and I3COH formation, and by measuring the
1
8
18
18
incorporation of O from
2 2
O and H O into I3COH. The
results demonstrate that (1) there is a high intramolecu-
lar deuterium isotope effect in the formation of 3MEI that
may be due to a rate-limiting step of either hydrogen
atom or aminium radical proton abstraction, (2) the
majority of I3COH is formed by hydration of 3MEI, and
(3) the ratio of dehydrogenation vs monooxygenation of
3
MI that occurs in goat lung microsomes is unusually
Intramolecular deuterium-isotope effects were determined by
high when compared with other P450 substrates that also
undergo dehydrogenation. Goat lung microsomes were
used for these studies because goats are one of the most
sensitive species to the toxic effects of 3MI.
incubating 0.7 mM 3MI-d
2
under the identical conditions
described above, except that the reactions were stopped after 8
min with 6.0 mL of ice-cold acetonitrile, and the total incubation
volumes were 6.0 mL. After the protein pellets were precipi-
+
+

3
-Methylindole Dehydrogenation
Chem. Res. Toxicol., Vol. 9, No. 1, 1996 293
dryness under a stream of N before derivatization with MSTFA/
tated, the supernatants were decanted and evaporated under a
stream of N until the organic solvent was removed. The
remaining aqueous solutions were extracted with Sep-Pak
cartridges (Waters Associates, Millford, MA). The Sep-Pak
cartridges were pre-wet with 15 mL of acetonitrile and then
rinsed with 15 mL of 0.05 M aqueous ammonium carbonate (pH
2
2
acetonitrile (vide supra) for analysis by GC/MS. The heavy-
isotope enrichment of water in the incubations was determined
by 18O incorporation into benzoic acid that was formed by
hydrolysis of benzoyl chloride. The benzoyl chloride was added
to aliquots that were removed from the incubations, rather than
to the incubations themselves, because I3COH is unstable under
6
.8). The samples were placed on the cartridges which were
1
8
then rinsed with 1.0 mL of 0.05 M aqueous ammonium carbon-
ate and then eluted with 5.0 mL of acetonitrile. The eluate was
stripped of solvent in vacuo then derivatized in 50 µL of a
MSTFA/acetonitrile (1:1) solution by heating to 80 °C for 30 min
in screw-cap sealed glass tubes.
2
acidic conditions. H O enrichment of the incubations was
determined individually to account for variations introduced
when unlabeled water, present in the microsomes, was added.
To each of the 50-µL aliquots that were removed prior to the
incubations was added 5 µL of benzoyl chloride, and then they
were immediately extracted three times with 50-µL portions of
ethyl acetate. The extracts from each aliquot were pooled,
In d ole-3-ca r bin ol Deh yd r a tion . Enzymatic and nonen-
zymatic dehydration of I3COH was determined by incubation
of I3COH in the presence of 8.0 mM NAC for 30 min at 37 °C
in 0.05 M potassium phosphate buffer (pH 7.4) under the
following conditions: (1) 0.6 mM I3COH alone; (2) 0.6 mM
I3COH, 1.0 mM NADPH, and goat lung microsomes; (3) 0.6 mM
I3COH and goat lung microsomes; and (4) 0.01 mM I3COH, 1.0
mM NADPH, and goat lung microsomes. The final P450
concentration in those incubations that included microsomes
was 1.1 nmol/mL. The samples were run in duplicate and were
prepared for analysis as described for determination of the
intermolecular isotope effect.
evaporated to dryness under a stream of N
with MSTFA for GC/MS analysis.
2
, and derivatized
Liqu id Ch r om a togr a p h y. HPLC analysis was performed
on a Beckman System Gold HPLC composed of a Model 126
solvent module and a Model 166 detector. Separation of
analytes was achieved on a 250 × 4.6 mm Ultramex C18, 5 µm
HPLC column (Phenomenex, Torrance, CA) with a gradient
solvent system that started at 20% 0.05 M ammonium acetate,
pH 6.0, in acetonitrile and was ramped to 95% over 10.0 min
and held there for 2.0 min before returning to 20% over 3.0 min.
The UV detector monitored the absorbance at 280 nm, the λmax
of the benzenoid bands of I3COH and 3MINAC, and the
metabolites were quantified by interpolation of their peak areas
with standard curves established on each day of the analyses.
Each sample was analyzed in triplicate.
18O
In cor p or a tion . Incorporation of 18O from molecular
2
oxygen was determined by incubating 1.0 mM 3MI under the
conditions described for the isotope effect experiments, except
that in one set of incubations there was no NAC, in a total
volume of 3.0 mL. The incubations were conducted in an air-
tight, four-flask glass manifold system constructed so that it
Ga s Ch r om a togr a p h y/Ma ss Sp ectr om etr y. Isotopic en-
richment of the deuterated 3MI substrates was determined with
a VG Micromass 7070H gas chromatograph/mass spectrometer
in the positive ion mode. Chromatography was performed on a
30 m wide bore DB-5 column (J &W Scientific, Folsom, CA) with
a head pressure of 5 psi and a temperature program that was
1
8
could be evacuated, flushed with N
%
2
, and have
2
O (50 atom
) introduced from a lecture bottle. The incubation apparatus
was generously loaned by Dr. William F. Trager, University
of Washington. 3MI was incubated with goat lung microsomes
in two of the flasks, and acetanilide was incubated with mouse
liver microsomes in the other two flasks. Mouse liver mi-
crosomes were required for the hydroxylation of acetanilide
because goat lung microsomes formed p-hydroxyacetanilide to
such a small extent that it would have been very difficult to
quantify the 18O incorporation. After placing all of the incuba-
tion ingredients except for substrate into the system, the flasks
were cooled on ice and the system was alternately evacuated
-
1
held at 100 °C for 1 min, ramped to 135 °C at 10 °C min , and
then held for 5 min before returning to 100 °C. The transfer
line, re-intrant, and injector temperatures were 250, 200, and
250 °C, respectively. 3MI undergoes facile loss of a 3-methyl
hydrogen under typical, 70 eV electron impact conditions. Such
a loss would interfere with the determination of deuterium
incorporation, so the ionization voltage was adjusted to 11 eV,
the point where no loss of hydrogen from unlabeled 3MI was
detected. In addition, the ion repeller was grounded to the
source block to further protect from imparting ion energy
sufficient to cause hydrogen loss. The ion abundance under
these conditions was poor but adequate for the intended
purpose. Ion channels corresponding to a mass range from M
and flushed with ultrapure N
%
2
at least 10 times. The 50 atom
O oxygen was introduced, substrate was added through
1
8
rubber septa, and the flasks were warmed to 37 °C and the
contents stirred during a 30 min incubation. The reactions were
stopped by immersing the flasks in a dry ice/acetone bath, and
the flasks were removed from the manifold system. Three
milliliters of ethyl acetate were placed on top of the frozen
incubation solutions, and as they thawed, they were mixed to
denature the protein and prevent any further metabolism in
the presence of atmospheric O . The ethyl acetate layer was
2
removed, and the aqueous solutions were extracted twice more
with 3.0-mL aliquots of ethyl acetate. The pooled organic
- 2 to M + 5 of 3MI-d
0
were monitored in SIM mode.
The ratios of metabolically produced isotopologs were deter-
mined by mass spectrometry with a Finnigan MAT 4500 gas
chromatograph/mass spectrometer. The GC was fitted with a
30 m DB-5 microbore column with 0.25 µm film thickness (J &W
Scientific). The carrier gas was hydrogen at a flow rate of ca.
-
1
extracts were evaporated to dryness under an N
ambient temperature and then derivatized in MSTFA/acetoni-
trile as described above.
2
stream at
30 m s . The GC oven temperature profile started at 100 °C
-
1
for 2.0 min and then was ramped at 10 °C min for 13.0 min
-
1
to 230 °C, then to 300 °C at 20 °C min , before returning to
100 °C. The injector, ionizer, and transfer line temperatures
were 270, 120, and 280 °C, respectively. Chemical ionization
of the deuterated compounds was achieved using methane/
ammonia (1:1) with (perfluorotributyl)amine enhancement (19),
and ionization of the derivatized 18O labeled I3COH, p-hydroxy-
acetanilide, and benzoic acid was accomplished by methane
chemical ionization. The spectrometer was operated in the
positive-ion mode, and each sample was analyzed in triplicate.
18O In cor p or a tion . Incorporation of 18O from H 18O into
H
2
2
I3COH was determined by incubating 3MI in a system in which
the water was enriched with H
prepared by diluting 0.70 mL of 0.12 M phosphate buffer (pH
7
1
8
2
O. Phosphate buffer was
1
8
2
.4) with 1.0 mL of H O (95 atom %) to a final buffer
concentration of 0.05 M. Duplicate incubations were set up by
adding 67 µL of goat lung microsomes (final P450 concentration
of 1.1 nmol/mL) to 433 µL of the buffer that had NADPH added
to a final concentration of 1.0 mM. 3MI was added in 5 µL of
ethanol, the flask contents were rapidly mixed, and 50-µL
aliquots were immediately removed from each flask and placed
in glass tubes on ice. The incubations were continued for 30
min at 37 °C and stopped by adding 500 µL of ice-cold ethyl
acetate. The organic layers were removed, and the samples
were extracted twice more with 500 µL of ethyl acetate. The
extracts from each incubation were pooled and evaporated to
A full-scan spectrum of 3MINAC(TMS)
standards ionized with CH /NH showed that both compounds
gave only one peak at m/ z 202 corresponding to [MH -
3 2
and I3COH(TMS)
4
3
+
+
N-acetylcysteine(TMS) ] and [MH - OTMS] for 3MINAC and
2
I3COH, respectively; thus, the fragments of labeled metabolites
would retain any deuterium atoms present in the compounds
before fragmentation. The ion current for each ion of interest
was enhanced during quantitation of the isotopolog ratios by
+
+

2
94 Chem. Res. Toxicol., Vol. 9, No. 1, 1996
Skiles and Yost
Ta ble 1. Kin etic Con sta n ts a n d Ca lcu la ted In ter - a n d In tr a m olecu la r Deu ter iu m Isotop e Effects
intermolecular
intramolecular
Dk
metabolite
substratea
Km (mM)
Vmax
DV
V/K
D(V/K)
3
MINAC
3MI-d0
0.061 ( 0.0018
0.020 ( 0.0005
8.7 ( 0.25
3.3
143 ( 4.2
133 ( 3.3
1.1
5.5 ( 0.5
3
MI-d3
3MI-d0
MI-d3
2.6 ( 0.065
1
3COH
0.10 ( 0.0042
0.029 ( 0.0013
1.3 ( 0.055
0.34 ( 0.15
3.9
13 ( 0.55
12 ( 0.53
1.1
4.8 ( 0.5
3
a
Substrate used in intermolecular deuterium isotope effect experiments. 3MI-d2 was the substrate used for the intramolecular isotope
effect experiments.
dividing the isotope effect, expressed as the ratios of the d
2
to
d
1
metabolites, by 0.5 because the d /d ratio would be 0.5 in
2
1
the absence of an isotope effect. Deuterium incorporation into
the labeled 3MI was sufficiently high that no further corrections
1
8
of the data were performed. The value for O incorporation
into I3COH from 18
18
2
O was corrected for the O incorporation
into p-hydroxyacetanilide. Incorporation of 18O into benzoic
acid, formed from hydrolysis of benzoyl chloride by the H
1
8
2
O
enriched water, served as the positive control and was used to
correct the values for 18O incorporation into I3COH from H
18
O.
2
Because oxygen exchange of carboxylic acids can occur in an
1
8
aqueous environment (22), incorporation of two atoms of
O
was evaluated in the analyses that measured 18O incorporation
into benzoic acid. The benzoic acid was extracted as rapidly as
possible to minimize exchange, and no detectable exchange
occurred as evidenced by the lack of any benzoic acid with two
atoms of 18O (data not shown).
Resu lts
F igu r e 1. Lineweaver-Burk plots showing kinetic deuterium
isotope effects on 3MINAC (A) and I3COH (B) formation from
In ter m olecu la r Deu ter iu m Isotop e Effects. The
quantities of 3MINAC and I3COH produced from the
various concentrations of 3MI used in the intermolecular
deuterium-isotope effect experiments were used to con-
struct the Lineweaver-Burk plots shown in Figure 1. In
all cases the amount of 3MINAC formed was about 10
times that of the I3COH. No other known metabolites
of 3MI were present to an appreciable extent (<5% of
the 3MINAC) in the time frame of the incubations.
The metabolism kinetics for the formation of 3MINAC
3
3
MI in goat lung microsomes. The substrates were unlabeled
MI (3MI-d ) and 3-( H -methyl)indole (3MI-d ). Error bars
0 2 3
3
indicate the standard deviations of three analyses of three
incubations at each concentration.
setting the mass spectrometer to a narrow scan width from m/ z
2
00 to 210.
Ionization of standard I3COH(TMS)
2
with CH
4
gave the
+
following spectrum [m/ z, ion (rel abundance)]: 320, [M + C
2
H
5
]
+
+
(18); 292, MH (63); 276, [M - CH
3
]
(76); 230 (18); 202, [M -
+
OTMS] (100). For quantitation of the isotopolog ratios the [M
]+ fragment was monitored because its abundance was
3
and I3COH from 3MI and 3MI-d were calculated from
-
CH
3
+
Figure 1 and are shown in Table 1. In the formation of
both compounds there was a decrease in Vmax upon
deuterium substitution that appears to indicate an
isotope effect of between 3 and 4. When the isotope effect
higher than that of MH , because it retained the oxygen atom,
and because it gave no M - 1 peak, which yielded more precise
calculations of the O incorporation. The spectrometer was set
to scan m/ z 274-286 to maximize the ion currents of each ion.
The CH
1
8
4
CI spectrum of standard p-hydroxyacetanilide(TMS)
2
+
on Vmax/K was compared, however, the apparent isotope
m
+
[
(
m/ z, ion (rel abundance): 324, [M + C H ] (24); 296, MH
2 5
effect on the pseudo-second-order rate constants was only
about 1.1. These results are consistent with a simple
+
100); 280, [M - CH
3
]
(94); 234 (19); 224 (10); 206, [M -
+
+
OTMS] (50)] indicated that monitoring the [M - CH
3
]
peak
model of enzyme kinetics described by Gillette (23) and
would give nearly the same ion current as the base peak and it
had virtually no M - 1 peak. The spectrometer was set to scan
m/ z 278-290 for quantitation of the isotopolog ratios. Ioniza-
tion of standard benzoic acid(TMS) yielded the following spec-
*
shown in eq 1, where ES is the substrate bound enzyme
k1
k2
k3
k4
E + S {k-1} ES {k-2} ES*
98 EP
98 E + P
(1)
trum: m/ z 223, [M + 29]+ (33); 195, MH+ (100); 179, [M - 15]
+
+
(51); 142 (15); 110 (15). The MH peak was monitored because
it was the base peak and because it had only a minor M - 1
peak. The spectrometer was set to scan m/ z 193-200 for
quantitation of the isotopolog ratios.
Ca lcu la tion s. For the intermolecular deuterium-isotope
experiments, the velocities of 3MINAC and I3COH production
with the activated perferryl-oxo complex and k
3
is the
isotopically sensitive step. Northrop (24), Gillette (23),
and others have shown that when there is a high
commitment to catalysis (k-2 ≈ 0), then Vmax is directly
proportional to k
3
(eq 2), and Vmax/K
m
is independent of
-
1
were calculated in terms of nmol of product (nmol of P450)
3
k (eq 3). The results of the intermolecular deuterium
-
1
min and plotted on a Lineweaver-Burk graph to determine
and Vmax. The overall catalytic efficiency (pseudo-second-
order rate constant) was calculated as k ) Vmax/K for each
product, and then the isotope effect was expressed in the
conventional manner as k /k
K
m
Vm
1
m
)
(2)
Et
1/k + 1/k + 1/k
2
3
4
H
D
.
Ratios of the deuterium and 18O isotopologs were determined
by the Brauman least-squares method (20) as further described
by Korzekwa et al. (21). For the intramolecular deuterium-
isotope experiments, the isotope effect was corrected for the
statistical probability of hydrogen or deuterium removal by
Vmax
E k k
t
1
2
)
(3)
Km
k2 + k
-1
isotope effect experiments shown in Table 1 are in
+
+

3
-Methylindole Dehydrogenation
Chem. Res. Toxicol., Vol. 9, No. 1, 1996 295
Ta ble 2. In cor p or a tion of 18O in to I3COH fr om 18O2 a n d
1
8
H2 O by Goa t Lu n g Micr osom es
18O incorporationa
(%)
exp.
1
18O source
18O2
product
measured
corrected
APAP
49.1 ( 0.33
6.65 ( 0.65
I3COHb
14
21
85
84
2
3
18O2
APAP
49.8 ( 1.1
10.3 ( 4.2
42.9d
I3COHc
H218
H218
O
O
BA
I3COH
36.6 ( 1.2
4
BA
I3COH
43.6 ( 0.95
36.7 ( 1.2
a
Percentage incorporated corrected for values of positive con-
1
8
trols: acetaminophen (APAP) for O2 in mouse liver microsomes
F igu r e 2. Time course of chemical and enzymatic dehydration
of I3COH as determined by the amounts of 3MINAC formed
from the methylene imine dehydration product. Incubation
conditions: (() 0.6 mM I3COH, (-) goat lung microsomes, (-)
NADPH; (9) 0.6 mM I3COH, (+) goat lung microsomes, (+)
NADPH; (0) 0.6 mM I3COH, (+) goat lung microsomes, (-)
NADPH; (b) 0.01 mM I3COH, (+) goat lung microsomes, (+)
NADPH. Details of the incubation conditions are described in
the Experimental Section.
and benzoic acid (BA) for H2 O incorporation. b N-Acetylcysteine
18
c
omitted from incubations. N-Acetylcysteine added to incubations.
Mean of two measurements.
d
lecular isotope-effect experiments. As seen in Figure 2,
incubation of a low concentration of I3COH produced
negligible amounts of 3MINAC when compared to the
total amount of 3MINAC produced during microsomal
incubation of 3MI. Moreover, in the intermolecular
isotope effect experiments, the concentration of 3MINAC
formed from 3MI was always about 10 times that of
I3COH, so the 3MINAC formed from the 0.01 mM
concentration of I3COH in the dehydration experiments
did not account for the large amounts of 3MINAC formed
from 3MI in the isotope effect experiments. Small
amounts of dehydration of I3COH occurred, but clearly,
chemical and enzymatic dehydration are insignificant
pathways of 3MEI formation.
agreement with this model and demonstrate that 3MI
dehydrogenation is catalyzed by P450 enzymes in goat
lung microsomes with a high commitment to catalysis,
m
masking the intrinsic isotope effect on Vmax/K .
In tr a m olecu la r Deu ter iu m Isotop e Effects. The
low substrate concentrations used in the intermolecular
isotope effect studies were necessary because the rate of
product formation was nonlinear at higher concentra-
tions. The substrate concentrations were significantly
1
8
2
O In cor p or a tion in In d ole-3-ca r bin ol. Incorpo-
m
below the apparent K s for 3MINAC and I3COH forma-
ration of molecular oxygen into I3COH, as determined
tion, which led to uncertainty about the validity of the
derived kinetic constants. Therefore, to verify an intrin-
sic kinetic deuterium isotope effect in the formation of
by its 18O content after incubation of 3MI in goat lung
1
8
microsomes under a 50 atom % O oxygen atmosphere,
is shown in Table 2. Surprisingly, the relative proportion
of I3COH formed by hydroxyl rebound in the absence of
NAC was quite small. Indeed, the majority (87%) of the
I3COH was formed by some other mechanism, probably
hydration of 3MEI. The relatively large amount of
I3COH formed by hydration of 3MEI in the absence of
NAC could plausibly be explained by a process in which
untrapped 3MEI reacted with water to an extent that
would not normally occur if the 3MEI had been trapped
3
MEI, the intramolecular deuterium isotope effect was
also determined. The intramolecular deuterium isotope
effect should not be susceptible to the masking observed
in the isotope effect on Vmax/K if there is free rotation
m
about the methyl carbon-carbon bond. The rotation
provides a mechanism that is effectively a branching
pathway that relieves the high commitment to the
incipient abstraction of a deuterium radical. The effect
of metabolic branching on unmasking of kinetic deute-
rium isotope effects has been previously investigated
experimentally by J ones et al. (25) and theoretically by
Korzekwa et al. (26). The results shown in Table 1
support the hypothesis developed above because an
intramolecular isotope effect of about 5 for I3COH
formation and 5.5 for 3MINAC formation was observed.
R-Secondary isotope effects were ignored in the calcula-
1
8
with a thiol. To explore this possibility, the O-
incorporation experiment was repeated with NAC added
to the microsomal incubation. In the presence of NAC,
approximately the same proportion of the I3COH (21%)
was formed by hydroxyl rebound (Table 2), indicating
that, even in the presence of NAC, most of the I3COH
(
80%) was formed by a different pathway.
1
8
H
2
O In cor por ation in to In dole-3-car bin ol. About
tions because they are typically small (k
H
/k
D
≈ 1.2) and
8
5% of I3COH in the absence of NAC was formed by a
they decrease the experimentally determined isotope
effect. Therefore, the value obtained represents a mini-
mum for the intrinsic isotope effect.
mechanism wherein oxygen that originated from water
was incorporated into the molecule (Table 2). These
1
8
results are in excellent agreement with the results of
O
Deh yd r a tion of In d ole-3-ca r bin ol. The formation
of 3MINAC from I3COH under various incubations
conditions is shown in Figure 2. Incubation of relatively
high concentrations (0.6 mM) of I3COH did not produce
significant amounts of 3MINAC under any of the condi-
tions tested. These experiments, however, did not ad-
dress the possibility that a goat lung microsomal enzyme
18
incorporation from
00% of the oxygen in I3COH could be accounted for
having originated from either O or H O. This is con-
2
O , also in the absence of NAC:
1
2
2
sistent with a hypothesis that most of the I3COH formed
from 3MI in goat lung microsomal incubations resulted
from hydration of the methylene imine.
with a very low K
m
may dehydrate I3COH to 3MEI;
Discu ssion
therefore, incubations were performed with a concentra-
tion of I3COH (0.01 mM) that more closely approximated
the amount of I3COH formed from 3MI in the intermo-
P450-supported dehydrogenation reactions are uncom-
mon, but the importance of the reactions is increasingly
+
+

2
96 Chem. Res. Toxicol., Vol. 9, No. 1, 1996
Skiles and Yost
2
recognized as they have been demonstrated to occur
during the metabolism of a number of compounds.
Valproic acid (27), ∆ -valproic acid (28), testosterone (29),
butylated hydroxytoluene (30), ethyl carbamate (31),
dihydropyridines (32), acetaminophen (33), flunisolide
the report on the dehydrogenation of ∆ -valproic acid, it
was noted that no monooxygenation products of allylic
rearrangement were found. This was presumably be-
cause the heme iron had access to the site of the second
electron abstraction but not the rearranged radical. In
the dehydrogenation of 3MI, the region of the substrate
in which the radical is resonance stabilized and from
where the second electron must be abstracted is the
same. Thus, the lack of any monooxygenation products
of benzylic rearrangement is not as easily explained.
Indeed, a monooxygenation at a 2-carbon radical may
occur, but the resulting 3-methylene hemiaminal (6)
would probably undergo rapid dehydration to 3MEI
(Scheme 1, pathway D).
2
(34), and lovastatin (35) all undergo net dehydrogena-
tions. The intermediacy of hydroxylated compounds that
dehydrate has been suggested to occur with several of
these compounds, and investigations have been con-
ducted to ascertain the mechanism of net dehydrogena-
tion. For example, early reports on the bioactivation of
acetaminophen suggested the possibility that an N-
hydroxylated intermediate dehydrated to form the N-
acetyl-p-quinone imine that reacted with GSH and was
believed to be at least partially responsible for covalent
binding and toxicity. Studies on the chemistry of syn-
thetic N-acetyl-N-hydroxy-p-aminophenol, however, dem-
onstrated that this compound was relatively stable and
was almost certainly not the precursor to the reactive
intermediate. Later work provided persuasive evidence
that two sequential one-electron oxidations by P450 was
the mechanism by which the N-acetyl-p-quinone imine
was formed (33).
Like other compounds that are dehydrogenated, mo-
nooxygenation at the site of hydrogen abstraction and
dehydrogenation of 3MI both occurred; however, unlike
many other examples, dehydrogenation of 3MI in goat
lung microsomes is the predominant reaction. Com-
pounds which can stabilize the intermediate by radical
resonance typically have relatively high partition ratios,
i.e., the ratio of dehydrogenation to monooxygenation. For
2
instance, ∆ -valproic acid had a partition ratio of about
The relatively high isotope effect observed in 3MI
dehydrogenation is uncommon for the P450-catalyzed
oxidation of a nitrogen-containing compound. While a
catalytic hydrogen-abstraction mechanism might explain
the isotope effect, reports that a high isotope effect can
occur in nonenzymatic proton transfers from an aminium
radical to a base (36) confound this interpretation. The
compounds that can be most closely compared with 3MI
are the dihydropyridines because they also lose an allylic
hydrogen in their P450-catalyzed dehydrogenation (15,
0.5 (28), and the ratio for testosterone is about 1 (29).
The ratios for other compounds such as valproate (27)
and ethyl carbamate (31), in which no allylic stabilization
of the radical is available, are 0.2 or less. The observed
partition ratio for 3MI was about 10; moreover, the 18O-
incorporation studies showed that the majority of the
I3COH was formed by hydration of 3MEI so that the
actual proportion of dehydrogenation exceeded that of
monooxygenation by over 50 times, a ratio that far
exceeds other substrates.
The mechanism of 3MI dehydrogenation to form 3MEI
most likely involves hydrogen atom abstraction as the
rate-limiting step, but a mechanism requiring rate-
limiting proton abstraction from an aminium cation
radical cannot be eliminated at this time. However, the
data presented here demonstrate that an unusually high
isotope effect in the reaction distinguishes 3MI from
similar nitrogen-containing substrates that also undergo
P450-catalyzed dehydrogenations. A small amount of
I3COH was shown to be formed by monooxygenation;
however, dehydrogenation dominated to a remarkable
extent, and the majority of I3COH was formed in goat
lung microsomes via hydration of 3MEI. Indeed, the
specificity of dehydrogenation rather than hydroxylation
of 3MI represents an unique case of preferential P450-
mediated dehydrogenation of a substrate to form a toxic
electrophilic intermediate.
3
2). Deprotonation of dihydropyridine aminium radicals,
formed by cytochrome P450 enzymes and peroxidases,
has recently been reviewed (37). The pK
of the dihy-
a
dropyridine aminium radical was estimated to be ca. 3.5,
a value similar to that reported for the 3MI aminium
radical of 5.0 ( 0.1 (13). If 3MEI formation is initiated
by N-oxidation, then like the dihydropyridines, specific
+
[
Fe-O]2 proton transfer is unlikely to occur because the
acidic γ proton would be inaccessible when the substrate
is oriented in the enzyme for oxidation of the nitrogen
(15).
Equally probable, and perhaps more compelling be-
cause of its simplicity, is a mechanism in which P450-
catalyzed hydrogen abstraction from the 3-methyl posi-
tion occurs in lieu of nitrogen oxidation because of the
orientation of the substrate in the binding site. Recent
findings (38) indicate that amine dealkylations can
indeed proceed via initial hydrogen atom abstraction,
although it is uncertain whether these findings extend
to the case of hydrogens that are γ to the amine nitrogen.
Stabilization of a radical metabolic intermediate is an
emerging mechanistic explanation that can, in some
cases, account for the occurrence of a dehydrogenation
reaction in lieu of monooxygenation. In a report on the
Ack n ow led gm en t. This work was supported by
United States Public Health Service Grant HL13645 from
the National Heart, Lung and Blood Institute and by a
United States Public Health Service Research Career
Development Award (Grant HL02119) to G.S.Y. We are
grateful to Drs. Douglas E. Rollins and Roger L. Foltz,
of the Center for Human Toxicology, and Mr. William
Howald, of the University of Washington Department of
Medicinal Chemistry, for providing mass spectrometry
instrumentation.
2
dehydrogenation of ∆ -valproic acid (28), resonance sta-
bilization of the allylic radical was invoked to explain the
relatively high dehydrogenation to monooxygenation
ratio, the “partition ratio”. That same reasoning can be
extended to the case of 3MI dehydrogenation initiated
by hydrogen radical abstraction. The benzylic radical
intermediate formed by hydrogen atom abstraction from
the methyl group should be even more stable than an
allylic radical and provide a greater opportunity for
abstraction of the second electron from the substrate. In
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TX9501187
+
+