Oxidation of 2,6-dimethylaniline by recombinant human cytochrome P450s and human liver microsomes

Article abstract of DOI:10.1021/tx000181i

2,6-Dimethylaniline (2,6-DMA) is classified as a rodent nasal cavity carcinogen and a possible human carcinogen. The major metabolite of 2,6-DMA in rats and dogs is 4-amino-3,5-dimethylphenol (DMAP) but oxidization of the amino group to produce metabolite

Full text of DOI:10.1021/tx000181i

672
Chem. Res. Toxicol. 2001, 14, 672-677
Oxid a tion of 2,6-Dim eth yla n ilin e by Recom bin a n t
Hu m a n Cytoch r om e P 450s a n d Hu m a n Liver Micr osom es
J inping Gan,† Paul L. Skipper,‡ and Steven R. Tannenbaum*,†,‡
Department of Chemistry and Division of Bioengineering and Environmental Health,
Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
Received August 21, 2000
2,6-Dimethylaniline (2,6-DMA) is classified as a rodent nasal cavity carcinogen and a possible
human carcinogen. The major metabolite of 2,6-DMA in rats and dogs is 4-amino-3,5-
dimethylphenol (DMAP) but oxidization of the amino group to produce metabolites such as
N-(2,6-dimethylphenyl)hydroxylamine (DMHA) is also indicated by the occurrence of hemo-
globin adducts of 2,6-DMA in human and rats. Previous studies have shown a large
interindividual variability in human 2,6-DMA hemoglobin adduct levels. In the present study,
2,6-DMA oxidation in vitro by human liver microsomes and recombinant human P450 enzymes
was investigated to assess whether the hemoglobin adduct variability could be attributed to
metabolic differences. At micromolar concentrations, the only product detectable (UV) was
DMAP, while at 10 nM, DMHA was a substantial product. 2E1 and 2A6 were identified as the
major P450s in human liver microsomes responsible for the production of DMAP by using
P450-specific chemical inhibitors and mouse monoclonal antibodies that selectively inhibit
human P450 2E1 and 2A6. 2A6 was identified as the major P450 responsible for the
N-hydroxylation. Native P450 2E1 and human liver microsomes catalyzed the rearrangement
of DMHA to DMAP independent of NADPH. Consistent with a mechanism involving oxygen
rebound to the heme iron center, labeled oxygen was not incorporated into DMAP from either
18O2 gas or H2 18O in this rearrangement. Results presented here suggest much of the observed
interindividual variability of 2,6-DMA hemoglobin adduct levels could be due to differences in
the relative amounts of hepatic 2E1 and 2A6.
hemoglobin adduct levels of 2,6-DMA, and this variation
is not related to cigarette smoking (8). Thus it appears
that there is a wider variation in exposure to this amine
than to many others that have been investigated or that
there exist exceptionally large interindividual differences
in its metabolism and disposition.
In tr od u ction
2,6-DMA1 is an industrial intermediate used in the
production of dyes, pesticides, and pharmaceuticals (1).
Several local anesthetic drugs, such as lidocaine and
ripovacaine, are made with 2,6-DMA, and one possible
source of exposure to 2,6-DMA may be these drugs since
2,6-DMA and DMAP are, for example, the major me-
tabolites of lidocaine both in vivo and in vitro (2-4). 2,6-
DMA is a rat nasal cavity carcinogen and a possible
human carcinogen (1, 5). How 2,6-DMA exerts its carci-
nogenic effect is currently unclear but a common mech-
anism for the carcinogenicity of many aromatic amines
is N-oxidation by P450s followed by phase II conjugation
of the resulting N-hydroxylamine to yield intermediates
that react with DNA to form genotoxic DNA adducts (6).
Consistent with this mechanism, it has been shown that
O-acetylated DMHA is capable of forming DNA adducts
in vitro (7).
The N-hydroxylation product of 2,6-DMA, DMHA, is
isomeric with 4-amino-3,5-dimethylphenol (DMAP). In
vivo study of 2,6-DMA metabolism in rats and dogs
demonstrated that DMAP and its conjugates are the
major metabolites in the urine of both species (9). The
aminophenol may be formed directly by oxidation at the
para position of the aromatic ring. The hydroxylamine
might also be a source of DMAP. DMHA is known to
rearrange to DMAP, at least under mildly acidic condi-
tions, in a reaction known as the Bamberger rearrange-
ment (10, 11). DMAP is also of interest because it is
potentially genotoxic by a mechanism involving nonen-
zymatic oxidation to the iminoquinone and quinone which
are strongly electrophilic species (12-14). A diagram of
proposed 2,6-DMA biotransformation is shown in Figure
1.
N-Hydroxylarylamines react with and covalently bind
to hemoglobin, and the adducts formed could serve as
biomarkers of exposure of the corresponding arylamines.
There is exceptional interindividual variability in the
P450s comprise a large family of hemoproteins involved
in the activation and detoxification of numerous drugs,
environmental pollutants, procarcinogens, and endog-
enous substances. To date, more than a dozen human
P450s have been successfully cloned and expressed in
various host cells, thereby enabling the identification of
individual P450s responsible for the metabolism of
selected xenobiotics through a combination of experi-
* To whom correspondence should be addressed, Massachusetts
Institute of Technology, 56-731A, 77 Massachusetts Avenue, Cam-
bridge, MA 02139. Phone: (617) 253-3729. Fax: (617) 252-1787.
E-mail: srt@mit.edu.
† Department of Chemistry.
‡ Division of Bioengineering and Environmental Health.
1
Abbreviations: 2,6-DMA, 2,6-dimethylaniline; DMAP, 4-amino-
3,5-dimethylphenol; DMHA, N-(2,6-dimethylphenyl)hydroxylamine;
4-MP, 4-methylpyrazole; KTZ, ketoconazole.
10.1021/tx000181i CCC: $20.00 © 2001 American Chemical Society
Published on Web 05/17/2001

Oxidation of 2,6-Dimethylaniline
Chem. Res. Toxicol., Vol. 14, No. 6, 2001 673
Ch r om a togr a p h y a n d In str u m en ta tion . 2,6-DMA and its
metabolites were analyzed with a Hewlett-Packard 1090 HPLC
system and quantified by calibration curves using synthetic
standards. Separations were performed on a 5 µm Supelco LC-
ABZ column (4.6 mm × 25 cm) at a flow rate of 1.0 mL/min.
The mobile phase composition began at 20% methanol/80% 20
mM phosphate buffer (pH 6.8) for 5 min, increased to 40%
methanol at 15 min, and then to 70% methanol at 18 min. The
gradient was kept at 70% for an additional 7 min for a total
analysis time of 25 min. UV absorbance at 230 nm was used
for detection. DMAP, DMHA, and 2,6-DMA eluted at 8, 15, and
20, respectively. Mass spectrometry was performed with a
Hewlett-Packard 5989A instrument. Samples were introduced
by gas chromatography using a Hewlett-Packard 5890 chro-
matograph with an HP-wax column (15 m × 0.25 mm). The MS
was operated in full scan mode to acquire spectra of DMAP. It
was operated in selected ion monitoring mode for analyses to
determine 18O incorporation, which was calculated from the
ratio of integrated peak areas at [M + 2]+ and (M+ + [M + 2]+).
En zym e Assa ys. Microsomal incubations were conducted at
37 °C in 100 mM phosphate buffer (pH 7.4; total volume 0.2
mL) containing an NADPH regeneration system (1.3 mM
NADP+, 3.3 mM glucose 6-phosphate, 3.3 mM MgCl2, and 0.1
unit of glucose 6-phosphate dehydrogenase). Microsomal protein
concentration was 0.5 mg/mL in all incubations. Reactants were
mixed in the order required for the different types of assays as
described below. Assays were terminated by addition of 50 µL
cold acetonitrile. After centrifugation, the supernatants were
stored at -80 °C prior to analysis.
F igu r e 1. Proposed oxidation and activation pathway for 2,6-
DMA. Oxidation of 2,6-DMA forms DMHA and DMAP. Phase
II conjugation of DMHA produces reactive esters that decompose
to a reactive nitrenium ion which can react with DNA and other
macromolecules. Reaction of the nitrenium ion with H2O is a
second pathway for the formation of DMAP. Further oxidation
of DMAP produces the toxic iminoquinone shown in the lower
left part of the figure.
ments using cDNA-expressed P450 enzymes and inhibi-
tory antibodies, as well as P450-specific inhibitors (15,
16). In the present study, we used this approach to
characterize the P450-catalyzed oxidation of 2,6-DMA in
order to determine the role of the oxidation pathway in
the genotoxicity of 2,6-DMA.
Oxidation of 2,6-DMA in the absence of inhibitors was
performed by adding enzyme to a mixture of all other compo-
nents already at 37 °C. 2,6-DMA was introduced in solution in
pH 7.4 phosphate buffer. The concentration of 2,6-DMA was
constant at 30 µM and incubations were conducted for up to 40
min.
Ma ter ia ls a n d Meth od s
Ch em ica ls a n d En zym es. Glucose 6-phosphate dehydro-
genase (Type VII) and all chemicals were purchased from Sigma-
Aldrich (St. Louis, MO). Recombinant human P450 1A2, 2A6,
2B6, 2C9, 2D6, 2E1, 3A4, FMO1, and FMO3, human lympho-
blast-control microsomes, human liver microsomes (cat. no.
H003), MAB2A6, and MAB2E1 were purchased from Gentest
(Woburn, MA). According to the vendor’s description, recombi-
nant P450 microsomes were expressed from their corresponding
transfected human cDNAs in a human lymphoblast cell line,
with P450 reductase coexpressed. 18O2 gas with a minimum 97%
isotopic abundance was obtained from Isotec (Miamisburg, OH);
H2 18O with 95% isotopic abundance was obtained from Aldrich.
DMHA was obtained from 2,6-dimethylnitrobenzene by re-
duction with Zn/NH4Cl according to the procedure for synthesis
of phenylhydroxylamine (17). The product exhibited the follow-
ing characteristics: UV (pH 7) λmax 238 nm; mp 90-92 °C [lit.
Chemical inhibition of 2,6-DMA oxidation was conducted by
preincubating liver microsomes with selected inhibitors at
various concentrations. The concentration range of the inhibitors
used were as follows: coumarin, 5-500 µM; ketoconazole (KTZ),
0.05-5 µM; orphenadrine, 10-500 µM; and 4-MP, 0.1-100 µM.
The P450-specific inhibitors, KTZ and 4-methylpyrazole (4-MP),
were preincubated with microsomes for 10 min at room tem-
perature. After the addition of NADPH, the reaction was started
by the addition of 2,6-DMA. The mechanism-based inhibitor
orphenadrine was preincubated with microsomes and NADPH
regeneration system for 15 min at 37 °C before the reaction was
started with the addition of 2,6-DMA. The competitive inhibitor
coumarin was added at the same time with 2,6-DMA. All
reactions were terminated with cold acetonitrile after 20 min
of incubation at 37 °C.
1
(18) mp 99 °C]; H NMR [(2H3C)2CO] δ 2.35 (s, 6 H, CH3), 6.98
Antibody inhibition experiments were conducted by preincu-
bating liver microsomes (5 µL) with solutions of antibody (10
µL) in Tris buffer (25 mM, pH 7.5) or Tris buffer itself (control)
on ice for 15 min. Antibody solutions were prepared by dilution
of the original solution supplied (10 mg of protein/mL) with Tris
buffer. The microsome-antibody mixtures were warmed to 37
°C and the reactions were started by addition of 30 µM 2,6-
DMA in phosphate buffer containing the NADPH regeneration
system. Incubations were terminated after 20 min at 37 °C.
Rearrangement of DMHA to DMAP was investigated by
adding cold microsomes to a solution of DMHA (50 µM) in
phosphate buffer at 37 °C without the NADPH regeneration
system. Inhibition of P450 2E1 rearrangement activity by 4-MP
and MAB2E1 was investigated as described for 2,6-DMA
oxidation, except that no NADPH regeneration system was
added.
(m, 3 H, aromatic), 7.36 (s, 1 H, OH) ppm [lit. (2) δ 2.35 (methyl),
6.50 (NH and OH), 7.05 (aromatics) ppm]. DMAP was synthe-
sized in two steps by coupling 3,5-dimethylphenol with the
diazonium ion formed by diazotization of sulfanilic acid and
reduction of the resulting azo dye with sodium hydrosulfite (19).
UV (pH 7) λmax 232, 294 nm; mp 180-183 °C [lit. (2) mp 179-
181 °C]; 1H NMR [(2H3C)2CO] δ 2.06 (s, 6 H, CH3), 3.62 (s, 2 H,
NH2), 6.38 (s, 2 H, aromatic), 7.20 (s, 1 H, OH) ppm [lit. (2) δ
2.06 (methyl), 4.22 (NH2), 6.47 (OH), 6.59 (aromatics) ppm].
[2-Methyl-3H]-2,6-dimethylaniline was custom synthesized by
catalytic dehalogenation of 2-amino-3-methylbenzyl bromide by
Moravek Biochemicals (Brea, CA). 2-Amino-3-methylbenzyl
bromide was synthesized by reacting 2-amino-3-methylbenzyl
alcohol with concentrated hydrobromic acid (20). Tritium labeled
2,6-DMA was purified from the dehalogenation mixture by silica
gel and HPLC, and had a specific activity of 2.5 Ci/mmol. The
tritiated 2,6-DMA was not fully characterized due to the limited
amount of material, but it has the same UV profile and same
HPLC retention time as the cold 2,6-DMA, and the position of
tritium label can be inferred to be at the methyl group from
the retention of radioactivity in the oxidation product after
microsomal incubation as described in the results section.
En zym e Assa ys w ith 18O2 a n d H218O. Experiments with
H218O were conducted essentially as described above using P450
2E1. Isotopically labeled H2O was diluted to 40% in the final
mixture. Incubations with 18O2 gas utilized a glass manifold
system (21). The system consisted of connections to argon gas,
vacuum, 18O2 gas source, and four outlets (one control, two

674 Chem. Res. Toxicol., Vol. 14, No. 6, 2001
Gan et al.
there was detectable formation of DMHA, which com-
prised 2-3% of the total. 2A6 was the only P450 that
exhibited N-hydroxylation activity, and DMHA produc-
tion by 2A6 was nearly 10% of DMAP production; 2E1,
along with other recombinant P450s tested, exhibited no
detectable activity. With respect to DMAP formation, as
at higher concentrations, 2E1 and 2A6 had comparable
activity. P450 2B6 exhibited no detectable activity for
formation of either product.
Ch em ica l In h ibition Exp er im en ts. 2,6-DMA (30
µM) was incubated with human liver microsomes in the
presence and absence of chemical inhibitiors. The effects
of KTZ, coumarin, 4-MP, and orphenadrine on the
p-hydroxylation of 2,6-DMA in liver microsomes are
shown in Figure 3. Coumarin was the most effective
inhibitor. Inhibition (50%) was achieved with ca. 30 µM
coumarin, and maximal inhibition of ca. 90% occurred
at concentrations greater than ca. 100 µM. 4-MP was also
an effective inhibitor, with only 10 µM 4-MP necessary
to achieve 50% inhibition. In contrast to coumarin,
though, 4-MP produced a maximal inhibition of only
about 60%. The other inhibitors tested were either
ineffective (KTZ) or produced only modest inhibition
(orphenadrine, ∼30%).
F igu r e 2. 2,6-DMA para-hydroxylation and N-hydroxylation
activities at 10 nM in human liver microsomes and recombinant
P450s. Data shown are the mean of two determinations and
corrected for background counts. The concentration values were
converted from the corresponding DPM values assuming DMAP
and DMHA produced had the same specific activity of 2.5 nCi/
pmol as [2-methyl-3H]-2,6-DMA.
samples, and one for NADP+ or DMHA solution) connecting the
manifold to the septum-capped reaction vials by needle. After
10 cycles of alternate evacuation and argon purging, 18O2 gas
was introduced; the vials were removed from the manifold and
the reactions were started by the addition of NADP+ (2,6-DMA
and acetanilide oxidation) or DMHA (DMHA rearrangement)
to start the reaction. To control for the presence of unlabeled
O2, experiments with acetanilide as substrate for hydroxylation
catalyzed by human liver microsomes were performed under the
same conditions (22). The reactions were terminated after 20
min of incubation by the addition of ethyl acetate. The organic
layer was collected after vortex and analyzed by GC-MS.
Kin etics. Reaction rates for 2,6-DMA oxidation to DMAP
were measured at 2,6-DMA concentrations of 10, 15, 30, 60, 120,
and 240 µM. DMHA concentrations of 25, 50, 100, 200, 400, and
800 µM were used to measure the rate of rearrangement of
DMHA to DMAP. Three time points (5, 10, and 15 min) were
used for each rate determination. The kinetic parameters were
calculated by nonlinear regression of the Michaelis-Menten
equation using GraphPad PRISM.
An tibod y In h ibition Resu lts. MAB2E1 produced a
maximal inhibition of 2,6-DMA oxidation activity of
human liver microsomes of 40%. MAB2A6 had a similar
inhibitory effect. When both MAB2E1 and MAB2A6 were
used, an inhibition over 90% was observed (Figure 4).
Kin etic P a r a m eter s of 2,6-DMA Oxid a tion . The
apparent KM values for P450 2E1, and liver microsomes
are very similar at 31 and 24 µM, respectively, while 2A6
has a slightly higher KM at 46 µM. The Vmax of 2E1 and
liver microsomes are almost identical at 1.43 and 1.45
nmole/min/mg protein; 2A6 has ∼3-fold lower Vmax at 0.40
nmol/min/mg of protein.
DMHA Rea r r a n gem en t. Incubation of DMHA with
P450 2E1 and liver microsomes resulted in the catalytic
rearrangement of DMHA to DMAP. Vmax of rearrange-
ment for P450 2E1 is 6.9 nmol/min/mg of protein, and
KM is 500 µM. The presence of an NADPH regeneration
system had no measurable impact on the reaction rate.
Heat denatured P450 2E1 (90 °C for 2 min) lost the
rearrangement activity completely.
En zym e Assa y w ith [2-m eth yl-3H]-2,6-DMA. [2-methyl-
3H]-2,6-DMA (10 nM, 2.5 µCi/nmol) was incubated with human
liver microsomes, P450 1A2, 2A6, 3A4, 1B1, 2B6, 2E1, and 2C9
for 20 min as described. The reaction products were separated
by HPLC, and the fractions coeluted with cold standard DMAP
and DMHA were collected and counted.
4-MP was effective in the inhibition of DMHA rear-
rangement activity of P450 2E1, with 60% inhibition at
20 µM. 4-MP achieved about 50% inhibition of the
rearrangement activity of human liver microsomes at the
same concentration. MAB2E1, however, had no effect on
either of the two systems (Figure 5). Incubations of
DMHA with P450 2A6, 1A2, and 3A4 yield much less
rearrangement activity than the incubations with P450
2E1 or liver microsomes (<5% of P450 2E1).
Oxygen La belin g Stu d ies. Reactions with 18O2 pro-
duced different results for 2,6-DMA oxidation and DMHA
rearrangement (Table 1). The incorporation of label from
18O2 into DMAP by P450 2E1 oxidation of 2,6-DMA was
quantitative. As a positive control, para-hydroxylation
of acetanilide by human liver microsomes under 18O2 had
a 98% of 18O incorporation (corrected for isotopic purity
of 18O2). Little, if any (0.8%), 18O label from 18O2 was
incorporated into DMAP in the DMHA rearrangement
experiment. DMHA rearrangement was also carried out
in H218O. Not more than 2% of label from H218O was
incorporated into DMAP.
Resu lts
2,6-DMA Oxid a tion P r od u cts. Standard mixtures of
2,6-DMA and the two isomeric monooxygenated products,
DMAP and DMHA were separated by HPLC using the
conditions described to provide a reference chromato-
gram. Synthetic DMHA was stable under the experiment
conditions. Incubation of 2,6-DMA (30 µM) with human
liver microsomes resulted in only one UV-observable peak
other than 2,6-DMA. This peak coeluted with authentic
synthetic standard DMAP, and its UV spectrum was
identical with the authentic standard spectrum. Incuba-
tion of 2,6-DMA with P450 2E1, 2A6, and 2B6 resulted
in the same peak. Incubations of 2,6-DMA with recom-
binant human P450 1A2, 3A4, 2C9*1, 2D6, FMO1, and
FMO3 yielded negligible amounts of DMAP. No produc-
tion of DMHA was observed by UV in any incubations.
A significantly different pattern was observed at low
(10 nM) concentrations of 2,6-DMA. First, under the
conditions used, nearly all substrate was converted to
products by liver microsomes as shown in Figure 2 and

Oxidation of 2,6-Dimethylaniline
Chem. Res. Toxicol., Vol. 14, No. 6, 2001 675
F igu r e 3. Inhibition of 2,6-DMA para-hydroxylation activity of human liver microsomes by various chemical inhibitors. Activities
are expressed as the percentage of DMAP formed in the absence of inhibitor.
F igu r e 4. Inhibition of 2,6-DMA para hydroxylation activity
of human liver microsomes by P450-specific inhibitory antibod-
ies. Activities are expressed as the percentage of DMAP formed
in the absence of antibody. The units of antibody concentration
are relative: 1.0 indicates that the antibody solution was used
as supplied without dilution.
Discu ssion
Cytochrome P450 functions as a monooxygenase by
catalyzing the cleavage of molecular oxygen and the
subsequent addition of an oxygen atom into the substrate.
In the case of 2,6-DMA, the nitrogen of the amino group
and the para position on the aromatic ring are the two
possible monooxidation sites leading directly to the
production of DMHA and DMAP, respectively. Incubation
F igu r e 5. Inhibition of DMHA rearrangement activity of P450
2E1 and human liver microsomes by 4-MP and MAB2E1.
of 2,6-DMA with recombinant P450 2E1, 2A6, 2B6, and
human liver microsomes produced DMAP as the only UV-
detectable product.
Activities are expressed as the percentage of DMAP formed in
the absence of 4-MP or antibody.
mal oxidation activity by ca. 90% at concentrations
greater than 100 µM. 4-MP, a P450 2E1-specific inhibitor,
reduced activity by 50% at concentrations as low as 10
µM. Orphenadrine, an inhibitor of 2D6, 2B6, and 2C9 at
concentrations lower than 500 µM (23), reduced the
activity by about 30%. Inhibition resulting from mouse
monoclonal antibodies raised against P450 2E1 and P450
To determine the P450(s) responsible for the para
hydroxylation, P450-specific inhibitors and P450-specific
inhibitory antibodies were used. KTZ, a P450 3A4-specific
inhibitor, failed to inhibit the microsomal oxidation
activity. On the other hand, coumarin, a substrate and
competitive inhibitor of P450 2A6 reduced the microso-

676 Chem. Res. Toxicol., Vol. 14, No. 6, 2001
Gan et al.
Ta ble 1. In cor p or a tion of 18O in to DMAP a
2A6 was the only P450 that exhibited this activity. 2E1
was inactive with respect to DMHA formation, but had
similar activity to that of 2A6 for production of DMAP.
Because 2E1 catalyzes not only oxidation of 2,6-DMA to
DMAP, but also the rearrangement of DMHA to DMAP,
the extent of DMHA production in liver could be highly
sensitive to the relative abundance of 2E1 and 2A6.
Considering that 2E1 is highly inducible and that 2A6
exhibits wide interindividual variability (31), it is reason-
able to expect that there would be large differences in
DMHA production among individuals after exposure to
2,6-DMA and that such metabolic differences may explain
much of the exceptional interindividual variability of 2,6-
DMA hemoglobin adduct levels.
DMHA induced mutations in Salmonella typhimurium
TA 100, and the O-acetylated DMHA formed dG adducts
at C-8 position (7). The autoxidation product of DMAP,
3,5-dimethyl-p-benzoquinone-4-imine, induced sister chro-
matid exchanges in cultured human lymphocytes (13).
Both types of these oxidative products of 2,6-DMA seem
to be of biological significance, but the relative impor-
tance of each in vivo is yet to be determined.
compd
source of 18
O
product
DMAP
DMAP
acetaminophen
DMAP
% isotopic
2,6-DMA
DMHA
acetanilide
DMHA
O2
O2
O2
H2O
100
0.8
98
2.0
a
The values in the table are corrected for the initial isotopic
abundance of 18O2 or H218O.
2A6 further indicates that P450 2E1 and 2A6 are the
major enzymes in human liver microsomes catalyzing the
oxidation of 2,6-DMA in vitro. P450 2E1, P450 2A6, and
human liver microsomes share similar KM values, and
either P450 2E1 or P450 2A6 could play the major role
in the oxidation of 2,6-DMA depending on the tissue
concentrations and interindividual polymorphisms of
these P450s.
In an attempt to elucidate the possible fate of DMHA,
we incubated DMHA with human liver microsomes as
well as other P450s. When DMHA was incubated with
P450 2E1 or human liver microsomes in the absence or
presence of NADPH, it was effectively rearranged to
DMAP. P450 2A6 also had detectable activity, but its
activity was much lower. Neither control microsomes
lacking P450 activity nor boiled P450 2E1 catalyzed
rearrangement of DMHA. Native enzyme structure thus
appears to be necessary for activity. 4-MP inhibited
DMHA rearrangement by both P450 2E1 and human
liver microsomes. Monoclonal antibody MAB2E1 had no
effect on rearrangement activity but effectively inhibited
oxidation of 2,6-DMA to DMAP. Rearrangement activity
was independent of whether an NADPH regeneration
system was present. Taken together, these results indi-
cate that binding of DMHA to a normal active site of P450
2E1 is required for rearrangement activity, which pro-
Ack n ow led gm en t. This work was supported by NIH
(Grant ES05622) and a DuPont educational grant.
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