0090-9556/97/2503-0384–389$02.00/0
DRUG METABOLISM AND DISPOSITION
Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics
Vol. 25, No. 3
Printed in U.S.A.
MECHANISM-BASED INACTIVATION OF MOUSE HEPATIC CYTOCHROME P4502B
ENZYMES BY AMINE METABOLITES OF MUSK XYLENE
LOIS D. LEHMAN-MCKEEMAN, DAVID R. JOHNSON1, DOUGLAS CAUDILL,
AND
SHARON B. STUARD
Human Safety Department, Corporate Professional and Regulatory Services, Procter and Gamble Co., Miami Valley Laboratories
(Received October 7, 1996; accepted December 16, 1996)
ABSTRACT:
the amine substitution relative to the t-butyl function. In the in vitro
studies with PB-induced mouse liver microsomes, both amines
inhibited PROD activity when preincubated in the absence of
NADPH. However, only p-NH2-MX caused a time- and NADPHdependent loss of PROD activity, and the inactivation rate was a
pseudo–first-order process that displayed saturation kinetics.
These results indicate that p-NH2-MX is a mechanism-based inactivator of mouse CYP2B enzymes. From kinetic analyses, the Ki
was calculated to be 10.5 mM and the kinact was 1.2 min21. As final
confirmation of the inhibitory effects of p-NH2-MX on mouse
CYP2B enzymes, the amine (0.67 mmol/kg) was dosed orally to
PB-induced mice. At 2 hr after dosing, p-NH2-MX inhibited essentially all of the PB-induced PROD activity, whereas an equimolar
dosage of parent MX had no effect at this early time. Thus, although MX is an inducer of mouse CYP2B enzymes, an amine
metabolite of MX is a mechanism-based inactivator of mouse
CYP2B10. Furthermore, it is likely that the amine is responsible for
the lack of functional CYP2B enzyme activity associated with induction of this enzyme by MX.
Cytochrome P450s constitute a superfamily of heme-containing
proteins that function in the biotransformation of a wide variety of
xenobiotics and endogenous chemicals. They are classified according
to structure into gene families and subfamilies, and the gene products
have been identified and isolated from a variety of plant and animal
species (1). Many of these enzyme families can be induced by
exposure to exogenous chemicals, a process usually, but not exclusively, mediated by transcriptional activation of the respective gene
(2, 3). The functional activity of these enzymes can also be inhibited
by a diverse group of chemicals and by a variety of biochemical
mechanisms (4 – 6).
The most specific inhibitors of cytochrome P450s are mechanismbased inactivators. These compounds are substrates for the target
enzyme that with metabolism are converted to an intermediate or
product that inactivates the enzyme. Inactivation is mediated by
covalent modification of a pyrrole nitrogen in the prosthetic heme
group or by direct modification of the heme moiety or apoprotein (4,
7, 8). This mode of inactivation is highly specific because the substrate must both bind to and be metabolized by the enzyme, and it is
readily distinguished from reversible inhibition, in which a substrate
must be present to exert the inhibitory effect, in that decreased enzyme
activity persists after the compound has been cleared from the body.
MX2 (fig. 1) is a synthetic nitromusk that has been widely used as
a perfume ingredient and fixative in a variety of household products.
Recent experiments have shown that MX treatment causes a PB-like
induction of mouse hepatic cytochrome P450 enzymes. Specifically, a
single dose of MX increased CYP2B10 mRNA in a manner similar to
PB and, with repeated dosing, MX treatment markedly increased liver
weight, caused hepatocellular hypertrophy, and increased immunoreactive CYP2B10 protein ;25-fold over control levels. Despite these
changes, however, there was no corresponding increase in CYP2B
enzyme activity associated with increased CYP2B mRNA and protein
(9, 10).
The lack of functional CYP2B activity suggested that MX, despite
inducing the enzyme, inhibited its catalytic function in some way.
When dosed to PB-induced mice, MX decreased CYP2B activity by
;90%, further confirming that MX inhibited mouse CYP2B activity.
However, in vitro experiments indicated that parent MX was not a
mechanism-based inactivator of the CYP2B enzymes (10).
In rodents, an important pathway in the metabolism of many
1
Present address: Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, Kansas City, KS.
D. R. J. was a Society of Toxicology Summer Intern.
Send reprint requests to: Dr. Lois D. Lehman-McKeeman, Procter and Gamble Co., Miami Valley Laboratories, P.O. Box 538707, Cincinnati, OH 45253-8707.
2
Abbreviations used are: MX, musk xylene; PB, phenobarbital; CYP2B, cytochrome P4502B; PROD, 7-pentoxyresorufin; p-NH2-MX, 4-amino-2,6-dinitro-1t-butylxylene metabolite; kinact, maximum rate constant for inactivation; o-NH2MX, 2-amino-4,6-dinitro-1-t-butylxylene metabolite.
384
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Musk xylene (2,4,6-trinitro-1-t-butylxylene; MX) is a synthetic nitromusk perfume ingredient that induces and inhibits mouse cytochrome P4502B (CYP2B) enzymes in vivo. The purpose of the
present work was to determine whether amine metabolites of MX
contributed to the enzyme inhibition and, if so, to define the nature
and kinetics of this inhibition. When dosed orally to phenobarbital
(PB)-treated mice, MX (200 mg/kg) inhibited >90% of the PBinduced O-dealkylation of 7-pentoxyresorufin (PROD), and [14C]MX
equivalents bound covalently to microsomal proteins. However,
when this experiment was repeated in mice pretreated with antibiotics to eliminate the gastrointestinal flora, no decrease in PBinduced PROD activity and no covalent binding to microsomal
proteins were observed. Thus, the ability of antibiotic treatment to
eliminate the enzyme inhibition and covalent binding implicated
amine metabolites of MX formed by nitroreduction in anaerobic
intestinal flora as obligatory for these effects. Two monoamine
metabolites of MX were synthesized to study enzyme inhibition
directly. These metabolites were 2-amino-4,6-dinitro-1-t-butylxylene and 4-amino-2,6-dinitro-1-t-butylxylene, referred to as
o-NH2-MX and p-NH2-MX, respectively, reflecting the position of
CYP2B INACTIVATION BY MUSK XYLENE METABOLITES
FIG. 1. Structures of MX and monoamine metabolites.
nitroaromatic compounds is reduction of one or more of the nitro
functions, yielding primary aromatic amines (11). In rats, nitroreduction of MX has been confirmed, with two monoamine metabolites
detected (12). This reductive metabolism is catalyzed largely by
nitroreductases in anaerobic bacteria residing in the gastrointestinal
tract and, as such, extrahepatic metabolism is an important contributor
to the overall fate of MX. In this regard, in vivo studies have suggested
that, in mice, nitroreduction of MX by anaerobic bacteria also occurs
and may be involved in the inhibition of CYP2B activity. Specifically,
when antibiotic treatment was used to eliminate the gastrointestinal
flora, MX no longer inhibited PB-induced CYP2B activity (10), thus
suggesting a role for the amine metabolites of MX in the inhibition of
CYP2B activity.
The major objective of the present work was to test the hypothesis
that monoamine metabolites of MX were central to the loss of CYP2B
enzyme activity in MX- and PB-induced mice. If observed, the second
objective was to characterize the nature and kinetics of this inhibitory
effect. To this end, monoamine metabolites of MX were synthesized
and their ability to inhibit and inactivate CYP2B enzymatic activity
was determined directly.
Materials and Methods
Chemicals and Reagents. MX was obtained from Polarome (Jersey City,
NJ) and was essentially 100% pure as determined by GC/MS analysis. [Methyl-14C]MX (18.2 mCi/mmol; synthesized by New England Nuclear, Boston,
MA) with a radiochemical purity of 97% as determined by HPLC-radiometric
analysis. Amine metabolites used in this work were synthesized by ironcatalyzed reduction of MX in the presence of hydrochloric acid and benzene
(12). The isomeric monoamine products were separated by preparative HPLC
on silica gel with a solvent system of hexane/ethyl acetate (90/10) as eluent.
Identity and purity of the monoamines were confirmed by GC/MS analysis.
PB (sodium salt), neomycin sulfate, tetracyline hydrochloride, bacitracin,
resorufin and 7-pentoxyresorufin were from Sigma Chemical Co. (St. Louis,
MO). A polyclonal antibody to rat CYP2B1 (Xenotech, Kansas City, KS) was
used to detect homologous mouse CYP2B proteins that were visualized with a
goat anti-rabbit IgG alkaline phosphatase conjugate (Bio-Rad, Hercules, CA).
All other reagents used for immunoblotting procedures were electrophoresis
grade.
Animals. Male B6C3F1 mice (Charles River Laboratories, Portage, MI;
20 –25 g) were used throughout. Animals were housed in humidity- and
temperature-controlled rooms and allowed free access to food (Purina Laboratory Rodent Chow, Ralston-Purina, St. Louis, MO) and water.
PB-induced microsomes used for in vitro experiments were prepared from
male B6C3F1 mice previously allowed ad libitum exposure to PB in drinking
water (0.05% as the sodium salt) for 5 days. At the end of this treatment period,
animals were fasted overnight before livers were removed, weighed, and
microsomes were prepared by differential centrifugation according to procedures outlined by Guengerich (13). Microsomes were pooled and stored at
280°C pending biochemical analyses.
Determination of CYP2B Enzyme Activity. O-Dealkylation of PROD,
used to assess CYP2B activity, was determined according to the fluorometric
method described by Burke et al. (14). Microsomal protein used in this assay
was typically 25–50 mg and, unless otherwise noted, the substrate concentration was 5 mM. Reactions were conducted at 37°C in fluorometric cuvettes
using a Perkin-Elmer LS50-B luminescence spectrometer (Norwalk, CT)
equipped with 4-position, thermostat-controlled, stirred cell holder. The assay
was calibrated with resorufin, and reaction rates, monitored over a 2-min
interval (excitation and emission wavelengths of 530 and 585, respectively),
were determined directly with the fluorescence data manager (Perkin-Elmer).
Microsomal protein levels were adjusted on an as-needed basis to ensure that
all reaction rates were linear (r2 . 0.95) over the 2-min data collection interval.
In Vitro Enzyme Inactivation Experiments. A two-stage incubation procedure was used to determine the ability of the amine metabolites to inactivate
CYP2B and to define the kinetics of the inactivation process. In the first stage,
the amines (2.5–15 mM) were incubated (37°C) with PB-induced microsomes
(5 mg/ml) and 200 mM phosphate buffer (pH 7.4) in the presence or absence
of an NADPH-generating system. The total volume of the first stage metabolism reaction was 1 ml, and amines were added in 1 ml of dimethylsulfoxide.
At 0.25, 0.5, 1, 1.5, 2, 3, 5, and 10 min, an aliquot was removed, diluted
100-fold into second-stage incubation (the PROD assay components as described previously), and the residual enzyme activity was determined.
In Vivo Analysis of Inhibition of CYP2B Enzyme Activity. Two experiments were conducted to evaluate the in vivo effects of MX on PB-induced
CYP2B enzyme activity. In the first experiment, the effect of antibiotic
treatment on CYP2B enzyme activity and covalent binding of [14C]MX equivalents to microsomal protein were determined. Four groups of mice (N 5
5/group) were exposed to PB in drinking water (0.05%) for 5 days and, during
this time, two groups were dosed twice daily (;7 a.m. and 5 p.m.) by gavage
with antibiotics (neomycin, tetracyline, and bacitracin at 400, 200, and 200
mg/kg/day, respectively), whereas the remaining mice received saline as
pretreatment control (10 ml/kg). On the afternoon of the fourth day of PB
treatment, mice were dosed by gavage with [14C]MX (0 or 200 mg/kg; 150
mCi/kg), and microsomes were prepared from fasted animals 18 hr after dosing
MX. PB and antibiotic treatment continued until necropsy.
In the second experiment, mice were allowed ad libitum exposure to PB in
drinking water (0.05% as the sodium salt) for 5 days, after which they received
a single oral dosage of MX or p-NH2-MX (0, 0.67 mmol/kg; structure shown
in fig. 1), and microsomes were prepared from food-fasted animals 2 or 18 hr
later (N 5 5/group). The 18-hr treatment groups were dosed with MX or
p-NH2-MX on the afternoon of the fourth day of PB treatment so that
microsomes from animals in the 2- or 18-hr groups were prepared on the same
day. From these samples, microsomal protein, total cytochrome P450 content,
PROD activity, and immunoreactive CYP2B protein levels were determined.
Other Biochemical Assays. To determine covalent binding, an aliquot of
microsomal protein from the PB-induced mice dosed with [14C]MX (2 mg;
;5000 dpm) was precipitated in 3 volumes of methanol. The precipitate was
washed and resuspended in water and precipitated two additional times with
methanol. After the third precipitation, 1 ml of 1 N NaOH was added and the
pellet was dissolved by heating at 60°C for 30 min. The dissolved protein
pellets were dialyzed overnight against a 3 kDa molecular cutoff membrane
(Pierce, Rockford, IL), after which aliquots were taken for liquid scintillation
counting (Packard, 2500 TR, Meriden, CT) and protein determination.
Microsomal protein was determined by the method of Bradford (15), with
bovine serum albumin as standard. The concentration of cytochrome P450 was
determined by the method of Omura and Sato (16), from the carbon monoxide
difference spectrum of dithionite-reduced microsomes with an extinction coefficient of 91 mM21 cm21.
Electrophoresis and Immunoblotting Procedures. Microsomal proteins
(5 mg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (17) on 7.5% polyacrylamide gels (5 3 10 cm, Mini-Protean gels,
Bio-Rad) and then transferred electrophoretically to nitrocellulose with a mini
trans-blot apparatus. The nitrocellulose was treated with blocker (5% nonfat
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 15, 2017
The structure of MX (2,4,6-trinitro-1-t-butylxylene) and the monoamine
metabolites used in this study. Amine metabolites were synthesized by ironcatalyzed acidic reduction of MX.
385
386
LEHMAN-MCKEEMAN ET AL.
TABLE 1
Effect of antibiotic treatment on inhibition of PROD activity and covalent
binding to microsomal proteins after administration of [14C]MX
Treatmenta Antibioticsb
Total P450
PROD
Covalent Binding
nmol/mg
pmol/min/mg
nmol [14C]MX/mg
PB/corn oil
2
1
1.94 6 0.16
1.74 6 0.11
1022 6 60
1098 6 67
PB/MX
2
1
1.86 6 0.04
1.86 6 0.07
92 6 16*
1355 6 13**
ND
ND
0.51 6 0.05
N/Dc
dried milk) for 1.5 hr and then incubated overnight (;16 hr) at 4°C in a 1/2500
dilution of anti-rat cytochrome P450 antibody diluted in blocker. For color
development, the nitrocellulose was rinsed in Tris-buffered saline before a 2-hr
incubation with the goat anti-rabbit IgG alkaline phosphastase conjugate
(1/1000 dilution in blocker) at room temperature. Immunoreactive protein was
visualized by the addition of nitroblue tetrazolium and 5-bromo-4-chloro-3indoyl phosphate as substrate for alkaline phosphatase. Blots were evaluated
by image analysis on a Macintosh Quadra 800 computer after scanning on a
Howtek Scanmaster Scanner (Howtek, Hudson, NH).
Data Analysis. Where appropriate, data were analyzed for statistical significance by analysis of variance, followed by Dunnett’s multiple comparison
test. Rate constants of inactivation were determined by linear regression
analysis of the natural logarithm of the residual activity as a function of the
preincubation time. All analyses were conducted with StatView statistical
application (Abacus Concepts, Berkeley, CA).
Results
MX is a trinitroaromatic compound, and the two monoamine metabolites synthesized and used in this work are shown in fig. 1.
Throughout this study, the 2-amino and 4-amino derivatives are
referred to as o-NH2- and p-NH2-MX, respectively; thus reflecting the
location of the amine substitution relative to the t-butyl function on
MX. The amine metabolites used in these studies were synthesized
and isolated with a purity of essentially 100%.
The major PB-inducible enzyme in mice is CYP2B10 (18). We
have previously shown that MX treatment inhibits CYP2B10 activity
in vivo (10). When [14C]MX was dosed to PB-induced mice, MX
reduced PROD activity by 90% of control values, and [14C]MX
equivalents were bound covalently to microsomal protein. However,
spectral determination of total cytochrome P450 levels was not affected (table 1). In contrast, with antibiotic treatment, PROD activity
in the PB-induced mice exposed to MX was increased relative to the
PB-induced level, and no covalent binding of [14C]MX equivalents to
microsomal proteins was detected. These results suggest that amine
metabolites of MX are responsible for both the covalent binding to
microsomal protein and inhibition of CYP2B enzyme activity.
To determine whether the monoamine metabolites inactivated
CYP2B activity, the compounds were preincubated with PB-induced
microsomes in the presence or absence of NADPH. In the absence of
Discussion
MX is a PB-like inducer of mouse cytochrome P450 enzymes.
However, despite increasing hepatic CYP2B10 mRNA and protein
levels, MX treatment does not increase CYP2B enzyme activity (9,
10). The results of the present studies indicate that when incubated
with PB-induced microsomes, p-NH2-MX caused a time- and
NADPH-dependent loss of PROD activity, and the inactivation rate
was a pseudo–first-order process that displayed saturation kinetics.
These characteristics are the hallmarks of mechanism-based inactivation (4, 19). When dosed to PB-induced mice, p-NH2-MX produced a
rapid, but prolonged loss of CYP2B activity. Collectively, these
results indicate that p-NH2-MX is a mechanism-based inactivator of
mouse CYP2B enzymes, and this inactivation is likely to explain the
absence of increased CYP2B activity in MX-treated mice.
A second monoamine metabolite, o-NH2-MX, was also evaluated
in these experiments. This metabolite inhibited CYP2B activity, but
did not inactivate the enzyme. With this compound, the amine function is adjacent to the t-butyl group, and it is possible that the bulky
t-butyl moiety provides sufficient steric hindrance to prevent or slow
the metabolism of this amine. Alternatively, these compounds may be
oriented in the substrate binding site of the CYP2B10 enzyme in a
manner that precludes oxidation of the amine substitution of o-NH2MX, while favoring oxidation of the p-NH2-MX metabolite. In either
case, the orientation of the amine substitution on MX is a major
determinant of the ability to inactivate the CYP2B enzyme. This
observation has important implications with respect to the possibility
that other nitromusk perfume ingredients may also inactivate cyto-
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Results represent the means 6 SE of 5 mice. ND, not determined; N/D, not
detected. p values are as follows: * statistically different from respective PB/
corn oil control ( p , 0.05); ** statistically different from respective PB/corn
oil control and PB/MX group not receiving antibiotics ( p , 0.05).
a
All mice were exposed to PB in drinking water (500 ppm) for 4 days and
then dosed with corn oil (10 ml/kg) or [14C]MX (200 mg/kg; 150 mCi/kg).
b
Mice were treated twice daily with a regimen of antibiotics (neomycin,
tetracycline, and bacitracin at 400, 200, and 200 mg/kg/day, respectively) for
the duration of the experiment. Control animals received saline on the same
dosing schedule.
c
Radioactivity levels were not above instrument background.
NADPH, both amines reduced PROD activity relative to control (fig.
2). However, although o-NH2-MX inhibited PROD activity, no further loss of enzyme activity was observed when preincubated with
NADPH, indicating that this metabolite does not inactivate CYP2B10.
In contrast, p-NH2-MX caused a time-dependent loss of PROD activity during preincubation with NADPH. The inactivation profile
exhibited a pseudo–first-order loss of activity, reaching maximum
inactivation at 5 min when ;85% of the enzyme activity had been
lost.
To define the kinetics of inactivation of PROD activity by p-NH2MX, the first-order rate constants for inactivation were determined
with various concentrations of the amine metabolite (table 2). The
double-reciprocal plot of the first order rate constants for inactivation
as a function of the inhibitor concentration is shown in fig. 3. From
this analysis, kinact was calculated to be 1.2 min21, and the concentration at which the rate constant for inactivation was half-maximal
(Ki) was 10.5 mM. The kinact of 1.2 min21 yields a maximum rate of
inactivation corresponding to a half-life of ;35 sec.
The potential for o-NH2-MX to inactivate the enzyme was determined up to a maximum concentration of 25 mM. This concentration
inhibited ;80% of the enzyme activity at zero time, but no change in
activity was determined in the presence of NADPH (results not
shown), thereby confirming the selectivity of the enzyme inactivation
for the p-NH2-MX metabolite.
As final confirmation that p-NH2-MX was responsible for CYP2B
enzyme inactivation, the amine metabolite was dosed to PB-induced
mice (fig. 4). At 2 hr after dosing, p-NH2-MX treatment eliminated
virtually all PROD activity. In contrast, MX treatment had no effect
on PROD activity at this time. At 18 hr after dosing, loss of PROD
activity was seen in both the p-NH2-MX- and MX-treated animals.
Despite the loss of enzyme activity, there was no loss of CYP2B
protein as determined by Western blotting (fig. 5) and no change in
the spectral detection of total cytochrome P450 content (results not
shown).
CYP2B INACTIVATION BY MUSK XYLENE METABOLITES
387
Liver microsomes were preincubated with 10 mM amine in the presence or absence of an NADPH-generating system for 0.25–10 min as described in Materials
and Methods, after which microsomes were diluted 100-fold and residual PROD activity was determined. The o-NH2-MX inhibited, but did not inactivate, the
enzyme, whereas the p-NH2-MX metabolite showed an NADPH-dependent, time-dependent loss of activity consistent with mechanism-based enzyme
inactivation. PROD activity in control microsomes [dimethylsulfoxide (DMSO) vehicle] was ;1100 pmol/min/mg. Results represent the mean of three separate
experiments.
TABLE 2
Inactivation of CYP2B10-dependent PROD activity by p-NH2-MX
p-NH2-MXa
Ki
21
t1/2
mM
min
min
2.5
5
7.5
10
15
0.234
0.377
0.471
0.636
0.725
2.96
1.84
1.47
1.09
0.96
a
Microsomes from PB-induced mice (5 mg/ml) were preincubated with the
concentrations of amine as described and an NADPH-generating system for
0.5–3 min. Aliquots of this reaction were removed at the specified time and
diluted 100-fold to determine PROD activity. Results were calculated from the
mean of three separate experiments.
chrome P450 enzymes. Specifically, MX is the only trinitrosubstituted
compound in this class, and no other member of the nitromusk class
(structures shown in ref. 20) possesses a nitro function in the orientation apparently needed for inactivation. Therefore, among the nitromusk perfume ingredients, it is likely that the inactivation of mouse
CYP2B10 is unique to MX and more specifically to the p-NH2-MX
metabolite of MX.
The inactivation of the CYP2B enzymes by p-NH2-MX was characterized by a Ki in the micromolar range and a high maximal rate
constant. Present data also suggest that p-NH2-MX likely inactivates
CYP2B10 by covalent modification of the apoprotein. MX or
p-NH2-MX treatment did not alter the spectral properties of cytochrome P450 enzymes or produce the characteristic green pigment
associated with porphyrin alkylation. Therefore, it is unlikely that
p-NH2-MX modifies the prosthetic heme group of the CYP2B10
enzyme by either metabolic intermediate complexation (21) or alkylation of a pyrrole nitrogen (22, 23). On the other hand, [14C]MX
equivalents bound covalently to microsomal proteins, and this binding
was abolished when nitroreduction was prevented by antibiotic treatment. Thus, although we do not have direct proof, the results of the
present studies indicate that covalent binding does occur, and because
FIG. 3. Double-reciprocal plot of the first order rate constant for
inactivation of CYP2B10 as a function of the concentration of p-NH2-MX.
PROD activity in PB-induced microsomes was determined at various times
after incubation with p-NH2-MX (2.5, 5, 7.5, 10, and 15 mM) as described in
Materials and Methods, and the first order rate constant for inactivation was
determined by regression analysis of the natural logarithm of the residual
activity as a function of the preincubation time. Only the linear portion of the
inactivation curve (0 –3 min) was used to determination the inactivation rate
constant, and these are noted in table 2. From this double-reciprocal plot, Ki
was determined from the intercept on the abscissa and kinact was calculated
from the intercept on the ordinate. Results were calculated from the mean of
three separate experiments.
the heme moiety is not altered, modification of the apoprotein is the
likely mode of inactivation. Although we have not identified the
nature of the reactive intermediate, N-oxidation to a reactive hydroxylamine intermediate (24, 25) may be the inactivating species.
A variety of chemical structures are known to be mechanism-based
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FIG. 2. Effect of o-NH2-MX and p-NH2-MX on inactivation of CYP2B10 in PB-induced mouse liver microsomes.
388
LEHMAN-MCKEEMAN ET AL.
MX or p-NH2-MX (0, 0.67 mmol/kg) were dosed by gavage to PB-induced
mice as described in Materials and Methods, and microsomes were prepared
from food-fasted animals at 2 or 18 hr after dosing the inhibitors. Control
animals were PB-induced and dosed with corn oil. p-NH2-MX treatment
almost completely eliminated PROD activity at 2 and 18 hr, whereas MX
treatment reduced PROD activity by ;90% of control values at 18 hr only.
Results represent the means 6 SE of 5 mice/group, and asterisks denote
statistical significance from the respective control ( p , 0.05).
FIG. 5. Hepatic levels of mouse CYP2B proteins after in vivo treatment of
MX and p-NH2-MX to PB-induced mice.
Microsomal samples (5 mg) from the experiment described in fig. 4 were
analyzed for CYP2B protein content by Western blotting. CYP2B proteins
were not affected by MX or p-NH2-MX treatment at 2 or 18 hr, despite causing
a significant loss of enzyme activity at one or both time points. A second,
smaller band of immunoreactive protein was observed in the MX-treated group
at 18 hr and in the p-NH2-MX–treated groups at 2 and 18 hr. The identity of
this band is not known at this time.
inactivators of cytochrome P450 enzymes (8), and several have been
shown to specifically or preferentially inactivate CYP2B enzymes in
different species. For example, chloramphenicol (26), N-methylcarbazole (27), secobarbital (28), phencyclidine (29), and acetylenes
[such as 9-ethynyl-phenanthrene (30) and N-aralkylated derivatives of
1-aminobenzotriazole (31)] are mechanism-based inactivators of
CYP2B enzymes. Although mouse liver microsomes were used in the
present studies, preliminary experiments suggest that p-NH2-MX also
inactivates rat CYP2B1 (unpublished data). Whereas a variety of
substituted aromatic amines have been shown to form metabolic
intermediate complexes with cytochrome P450 enzymes (32), the
identification of an aromatic amine with specificity to inactivate
Acknowledgments. We gratefully acknowledge the technical contributions of Dr. Ken Yelm and Mr. Harold Vaughn who synthesized
the monoamine metabolites of MX, and Dr. Pedro Rodriguez and Ms.
Cindy Eddy who completed the GC/MS analysis of these compounds.
References
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FIG. 4. Effect of in vivo treatment of MX and p-NH2-MX on PB-induced
PROD activity.
CYP2B enzymes represents a new chemical class of mechanismbased inactivators of this enzyme family. Additional studies will need
to be conducted to determine whether p-NH2-MX specifically inactivates CYP2B enzymes or whether it is capable of inactivating other
cytochrome P450 families.
It is well-established that nitroreduction to primary amines is a
major metabolic pathway for a variety of nitroaromatic compounds,
and the role of anaerobic intestinal bacteria in this reductive metabolism is recognized (11). In fact, intestinal bacteria play an obligatory
role in the metabolic activation and genetic toxicity of the hepatocarcinogen, 2,6-dinitrotoluene (33, 34). In contrast, despite the importance of bacterial nitroreduction in the enzyme inactivation seen with
MX, MX has been characterized as a nongenotoxic compound (35).
Therefore, despite similar routes of biotransformation, the fate of
amine metabolites of MX seems to be somewhat different from amine
metabolites of other nitroaromatic compounds.
This work was initiated because of the original observation that MX
induced a nonfunctional CYP2B10 enzyme. Based on the present
findings, it seems that MX is a direct inducer of CYP2B enzymes,
which, at the same time, can be metabolized to p-NH2-MX, a potent
inactivator of the induced enzymes. The complexity of this response
underscores the need to confirm the analysis of enzyme activity with
the evaluation of corresponding protein or mRNA levels. That is,
accurate determination of the effects of MX can only be achieved
when the enzyme activity data are compared with at least one additional level of enzyme regulation. In this regard, previous studies
characterizing the effects of MX on rat cytochrome P450 enzymes
have concluded, based on enzyme activity data alone, that MX had no
effect on CYP2B1 (20, 36 –38). Because p-NH2-MX is a major
metabolite in rats (12) and because preliminary data suggest that it
also inactivates CYP2B1, the possibility that MX can induce and
inactivate CYP2B enzymes in rats should be considered.
In summary, the results of the present study have identified an
amine metabolite of MX as a mechanism-based inactivator of mouse
CYP2B10, and it is likely that this amine is responsible for the lack of
functional CYP2B enzyme activity associated with induction of this
enzyme by MX. However, because of the specificity of the inactivation to the p-NH2-MX metabolite, it is unlikely that other, structurally
similar nitromusks will cause a similar inactivation. Collectively, the
results confirm that MX treatment will both induce and inactivate the
CYP2B enzymes.
CYP2B INACTIVATION BY MUSK XYLENE METABOLITES
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