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Vol. 285, Issue 3, 1287-1295, June 1998
Wadsworth Center, New York State Department of Health, Albany, New York (J.G., Q.Y.Z., T.W.L., X.D.); Department of Molecular and Cellular Physiology (M.B.G.) and Department of Environmental Health and Center for Environmental Genetics (D.W.N.), University of Cincinnati Medical Center, Cincinnati, Ohio; Pharmacogenetics Section, Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina (M.N.); and School of Public Health, State University of New York, Albany, New York (X.D.)
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Abstract |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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The metabolic activation of two known olfactory mucosal (OM) toxicants,
acetaminophen (AP) and 2,6-dichlorobenzonitrile (DCBN), was examined
with mouse liver and OM microsomes and purified, heterologously
expressed mouse CYP2A5 and CYP2G1. In reconstituted systems, both
isoforms were active in metabolizing DCBN and AP to metabolites that
formed protein adducts. The formation of DCBN- or AP-protein adducts
and other AP metabolites, including 3-hydroxy-AP and, in the presence
of glutathione, AP-glutathione conjugate, was also detected in OM
microsomal reactions and to a much greater extent than in liver
microsomes. Evidence was obtained that CYP2A5 and CYP2G1 play major
roles in mouse OM microsomal metabolic activation of DCBN and AP.
Immunoblot analysis indicated that CYP2A5 and CYP2G1 are abundant P450
isoforms in OM microsomes. OM microsomal AP and DCBN metabolic
activation was inhibited by 5- and 8-methoxsalen, which inhibit both
CYP2A5 and CYP2G1, and by an inhibitory anti-CYP2A5 antibody that also
inhibits CYP2G1. In addition, the roles of CYP1A2 and CYP2E1 in the OM
bioactivation of AP and DCBN were ruled out by comparing activities of
acetone-treated mice or Cyp1a2(
/) mice with those of
control mice. Thus, CYP2A5 and CYP2G1 may both contribute to the known
OM-selective toxicity of AP and DCBN. Further analysis of the kinetics
of AP and DCBN metabolism by the purified P450s suggested that CYP2A5
may play a greater role in OM microsomal metabolism of AP, whereas
their relative roles in DCBN metabolism may be dose dependent, with
CYP2G1 playing more important roles at low substrate concentrations.
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Introduction |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Numerous
environmental chemicals are known to cause tissue-selective toxicity in
the olfactory mucosa in rodents (Bonnefoi et al., 1991
; Dahl
and Hadley, 1991; Genter et al., 1992; Gu et al.,
1997). However, little is known about whether these compounds are also
toxic in human nasal mucosa. To better predict the potential toxicity
of environmental chemicals in humans and to determine the mechanism of
tissue-selective toxicity, we sought to identify the enzymes
responsible for their metabolic activation in the animal models
exhibiting toxicity.
In mice, several cytochrome P450 [P450; the nomenclature used in this
report is according to Nelson et al. (1996)] isoforms have
been detected in olfactory mucosa, including CYP1A2, CYP2A5, CYP2E1, CYP2G1 and one or more isoforms of the CYP3A
subfamily (Hua et al., 1997; Su et al., 1996; Gu
et al., 1997; Genter et al., 1998). Of these,
CYP2G1 is detected only in the olfactory mucosa (Hua et al.,
1996), whereas CYP2A5 is expressed in the olfactory mucosa at levels
much higher than in liver, kidney and lung (Su et al.,
1996). Heterologously expressed mouse CYP2G1 is active toward
testosterone and progesterone (Hua et al., 1997), which is
consistent with findings with rabbit CYP2G1 (Ding and Coon, 1994).
CYP2A5 is known to be the major coumarin 7-hydroxylase in mouse liver
(Negishi et al., 1989) and also has activity toward other
foreign chemicals, such as aflatoxin B1 and N-nitrosodiethylamine (Camus et al., 1993; Pelkonen et al., 1994).
In the present study, the roles were examined of CYP2A5 and CYP2G1 in
the metabolic activation of two known OM toxicants, DCBN and AP. DCBN
is a herbicide used for weed control. AP is a widely used therapeutic
agent. Both DCBN and AP have been shown to cause OM toxicity in mice
(Jeffery and Haschek, 1988; Brittebo, 1993; Deamer et al.,
1994). DCBN does not cause toxicity in liver at the doses that cause OM
toxicity (Brandt et al., 1990), whereas AP causes liver and
renal toxicity in addition to OM toxicity (Placke et al.,
1987; Jeffery and Haschek, 1988).
Metabolic activation of DCBN and AP was studied using purified P450s in
reconstituted systems and in liver and OM microsomal preparations.
Heterologously expressed mouse CYP2A5 and CYP2G1 were obtained and
purified to electrophoretic homogeneity. Antibodies, generated with
purified CYP2A5, and chemical inhibitors were used to demonstrate the
prominent roles of the two isoforms in OM microsomal metabolism. In
addition, the potential roles of CYP1A2 and CYP2E1 were ruled out in
experiments with animals lacking the Cyp1a2 gene (Liang
et al., 1996) or mice with elevated CYP2E1 levels after
acetone treatment. Furthermore, to determine the relative importance of
CYP2A5 and CYP2G1 in metabolic activation, the kinetics were examined
of AP and DCBN metabolism by the purified P450s. Our results indicate
that although both CYP2A5 and CYP2G1 are active toward DCBN and AP,
CYP2A5 may play a greater role in OM microsomal metabolism of AP,
whereas the relative roles of CYP2A5 and CYP2G1 in DCBN metabolism may
be dose dependent, with CYP2G1 playing a more important role at low
substrate concentrations.
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Materials and Methods |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Heterologous expression and purification of mouse CYP2A5 and
CYP2G1.
Baculovirus-mediated heterologous expression of mouse
CYP2G1 in insect cells has recently been achieved (Hua et
al., 1997). The bacterial expression vector for CYP2A5 was
constructed as described previously for CYP2A4 (Sueyoshi et
al., 1995). Escherichia coli cells expressing mouse
CYP2A5 were cultured in 1 liter of LB medium containing 100 µg
ampicillin, 0.2% glucose and 0.5 mM 
-aminolevulinic acid. The cells
were incubated in an orbital shaker at 37°C for 4 hr at 225 rpm
before addition of isopropyl-
-D-thiogalactoside (Stratagene, La Jolla, CA) to a final concentration of 0.5 mM and were
collected after a 48-hr incubation at 25°C and at 150 rpm. The cell
pellet from 1 liter of culture was washed in 40 ml of 20 mM potassium
phosphate buffer, pH 7.4, containing 50 mM KCl and 5 mM EDTA and
resuspended in 2 vol of the same buffer. The cells were lysed by three
passes through a French Pressure Cell (SLM) using the high setting and
a gauge reading of 600 to 700. Cell debris was removed by
centrifugation at 9500 rpm for 10 min in a JA 20 rotor, and the
supernatant was centrifuged again at 35,000 rpm in a Ti 45 rotor for 1 hr to obtain the membrane fraction. The membrane pellet was washed with
50 mM Tris-HCl buffer, pH 7.4, containing 0.8 M KCl and 1 mM EDTA, and
resuspended in 4 ml of 10 mM Tris-acetate buffer, pH 7.4, containing 1 mM EDTA and 20% glycerol, with a 15-ml Dounce all-glass homogenizer.
The membrane fractions from four 1-liter cultures were combined and solubilized by resuspending in 0.5% (w/v) Tergitol NP-10 and 1% (w/v)
sodium cholate in 0.1 M Tris-acetate buffer, pH 7.4, containing 0.15 M
KCl and 1 mM EDTA, with continuous mixing at 4°C for 2 hr. After
removal of insoluble material by centrifugation, the supernatant was
dialyzed overnight against 10 mM phosphate buffer, pH 7.7, containing 1 mM EDTA, 0.5% NP-10 and 20% glycerol (buffer A) and then loaded on a
50-ml DEAE-Sepharose column (Sigma Chemical, St. Louis, MO) previously
equilibrated with buffer A. CYP2A5 was recovered in the unbound
fraction, which was directly applied to a 10-ml HTP column (BioRad,
Hercules, CA) previously equilibrated with 10 mM phosphate buffer, pH
7.4, containing 0.5% NP-10 and 20% glycerol (buffer B). After washing
with 10-column vol of buffer B, the HTP column was eluted with a linear
gradient of potassium phosphate (10-300 mM, pH 7.4) in buffer B. A
homogeneous preparation of CYP2A5 was recovered at ~180 mM phosphate
buffer, which after dialysis overnight against buffer B, was applied to
a second HTP column (5 ml) to remove Tergitol NP-10 as described
previously (Ding and Coon, 1988). The bound CYP2A5 was eluted with 500 mM potassium phosphate buffer, pH 7.4, containing 0.5% sodium cholate and 20% glycerol. The final CYP2A5 preparation was dialyzed twice overnight against 2 liters of detergent-free buffer (50 mM potassium phosphate, pH 7.4, containing 20% glycerol and 0.1 mM EDTA) and stored
at 30°C.
). The
resulting 6% PEG supernatant was then diluted (1:2) with 20%
glycerol, and a 10% NP-10 stock solution was added to a final
concentration of 0.5%. The preparation (~80 nmol total P450) was
loaded onto a 30-ml HTP column previously equilibrated with buffer B
and eluted with a potassium phosphate buffer gradient (10-300 mM) as
described above. CYP2G1, which was recovered at ~150 mM phosphate,
was dialyzed overnight with 10 mM phosphate buffer, pH 6.4, containing
1 mM EDTA, 0.5% NP-10 and 20% glycerol (buffer C) and loaded onto an
8-ml S-Sepharose column (Sigma) previously equilibrated with buffer C. The column was washed with buffer C and eluted with a pH gradient from
6.4 (buffer C) to 7.4 (buffer B). A homogeneous preparation of CYP2G1
was recovered at pH ~7.0, which was dialyzed overnight with 10 mM
phosphate buffer, pH 7.4, containing 0.2% NP-10 and 20% glycerol and
applied to a HTP column (3 ml) to remove Tergitol NP-10. The bound
CYP2G1 was eluted with 500 mM potassium phosphate buffer, pH 7.4, containing 0.1% cholate, 0.1 mM EDTA and 20% glycerol. The final
CYP2G1 preparation was dialyzed against detergent-free buffer as
described above for the purification of CYP2A5.
Preparation of anti-CYP2A5 antibody and immunoblot analysis.
Polyclonal antibodies to CYP2A5 were prepared using purified CYP2A5 in
two female Flemish-Giant Chinchilla rabbits using a previously
described protocol (Ding and Coon, 1990). IgG fraction was prepared
from rabbit serum according to McKinney and Parkinson (1987).
Immunoblot analysis was performed with an ECL kit from Amersham as
described previously (Ding and Coon, 1990). The source of a monoclonal
antibody to rat CYP2E1 (mAb 1-98-1) and preparation of polyclonal
antibodies to rabbit CYP2A10/11 and CYP2G1 have been described in
earlier studies (Park et al., 1986; Ding and Coon, 1990;
Ding et al., 1991).
Determination of catalytic activities.
Formation of
DCBN-protein adduct and GSH conjugates of DCBN epoxide (GS-DCBN) was
assayed as described recently (Ding et al., 1996) with use
of 2,6-[ring-14C]DCBN (16.7 Ci/mol;
Sigma) as a substrate. The contents of reaction mixtures and the
incubation conditions are indicated in the legends to tables and
figures. Protein adducts were precipitated with ice-cold acetone,
washed repeatedly with sodium dodecyl sulfate (1%) to remove
nonspecific binding, dissolved in 1 M sodium hydroxide and analyzed by
liquid scintillation counting. The radioactivity in the washed
precipitates was compared with that of the total reaction mixture for
calculation of the rate of adduct formation. Formation of GS-DCBN was
determined by radiometric HPLC as described previously (Ding et
al., 1996).
with some modifications in the contents of reaction mixtures as indicated in the legends to tables and figures. Reactions were initiated by the addition of NADPH to a final volume of 250 µl
and were terminated after incubation at 37°C for up to 10 min by the
addition of 125 µl of 3 M perchloric acid. HPLC analyses of AP
metabolites were performed with a Waters µBondapak
C18 column according to Harvison et
al. (1988), with a radiometric HPLC system as described recently
(Ding et al., 1996). Metabolites were detected by absorbance
at 250 nm and by radioactivity measurements with 14C-AP as the substrate. Metabolites of AP were
identified on the basis of comigration with standards generated in
enzymatic reactions with rat CYP1A1 in a reconstituted system (data not
shown), which was previously shown to metabolize AP to 3-OH-AP and
GS-AP in the presence of added GSH (Harvison et al., 1988).
The retention times of the two metabolites were similar to the ones
reported earlier using the same chromatographic conditions (Harvison
et al., 1988). Formation of GS-AP was also confirmed by its
detection by radioactivity measurement in experiments with
3H-GSH and unlabeled AP (data not given).
Formation of AP-protein adduct was assayed as described above for DCBN
metabolism with use of
[ring-UL-14C]-AP (6.3 Ci/mol, from
Sigma) as a substrate.
Quantification of 3-OH-AP and GS-AP was initially carried out by
measuring radioactivity of individual peaks with an online radioactivity detector, and the amounts were calculated on the basis of
percent recovery of total radioactivity. Subsequently, the ratios of UV
absorbance at 250 nm to radioactivity of the two metabolites and that
of AP were calculated by plotting the integrated peak areas from UV
detector vs. those from radioactivity detector using samples
from >10 experiments. In all cases, the linearity of the plots
essentially extends through the origin (not shown). The slopes from
these plots (60.4, 44.3 and 81.4 µV*sec/cpm for AP, 3-OH-AP and
GS-AP, respectively; r > 0.99 for all three compounds) were used
to calculate the relative amounts of the two metabolites in reactions
with unlabeled AP as substrate in later experiments.
Other methods and materials.
Microsomes from combined liver
or olfactory mucosa of 6 to 10 mice or OM S9 fractions from individual
mice were prepared as described previously (Su et al., 1996;
Gu et al., 1997). Tissues from C57BL/6J mice were obtained
from a breeding stock maintained at the Wadsworth Center. Tissues from
Cyp1a2(/) mice and wild-type littermates were obtained
from breeding stocks maintained at the University of Cincinnati.
Preparation and initial characterization of the
Cyp1a2(/) mice have been reported previously (Liang
et al., 1996). Only males (~20-25 g b.wt.) were used. For
induction of CYP2E1, male C57BL/6J mice were given 1% acetone in tap
water in place of drinking water for 7 days, whereas the control groups received water only. Protein concentrations were determined by the
bicinchoninic acid method (Pierce, Rockford, IL) with bovine serum
albumin as the standard. P450 concentrations in microsomes or purified
preparations were determined according to the procedure of Omura and
Sato (1964). Absolute spectra of purified CYP2A5 and CYP2G1 were
recorded at room temperature using a Cary 3E spectrometer (Varian).
Purification of NADPH-P450 reductase and b5
from rabbit liver microsomes has been described in an earlier study
(Ding and Coon, 1994). Purified rat CYP1A1 was a gift from Dr. Laurence Kaminsky of the Wadsworth Center. Metyrapone and unlabeled AP were from
Sigma; 8-methoxsalen and 5-methoxsalen were from Aldrich. Other
materials were obtained as described previously (Ding and Coon, 1988,
1990; Peng et al., 1993; Ding et al., 1996).
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Results |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Spectral and electrophoretic characterization of purified,
heterologously expressed CYP2A5 and CYP2G1.
Bacteria-expressed
CYP2A5 and SF9 cell-expressed CYP2G1 proteins were purified to
electrophoretic homogeneity using conventional liquid-chromatographic
methods after detergent solubilization of membrane preparations. As
shown in figure 1A, a single band was
detected by silver stain in purified preparations of CYP2A5 (lane 1)
and CYP2G1 (lane 2). The two proteins migrated to about the same
position and were detected as a single band when mixed (not shown).
Efforts to resolve the two proteins by gel electrophoresis using
different concentrations of acrylamide and by varying the length of the
gels have so far been unsuccessful. For reasons not understood, mouse
CYP2G1 was not stained as intensely as CYP2A5 by silver, although the
two proteins were stained to a similar extent by Coomassie blue (data
not given); different amounts of protein were applied in figure 1 to
yield similar intensities. The specific contents of P450 in the
purified preparations were ~11.0 and ~6.8 nmol/mg protein for
CYP2A5 and CYP2G1, respectively, determined by CO-difference
spectroscopy using an extinction coefficient of 0.091 µM
1 cm1
(Omura and Sato, 1964). The reduced carbonyl difference spectra exhibited maximal absorbance at 449 nm for both cytochromes with no
peak at 420 nm (not shown) and absolute spectra of the cytochromes (fig. 1B) indicate that both are predominantly low spin in the ferric
form, with maximal absorbance at ~415 nm. The spin state of mouse
CYP2G1 in the ferric form contrasts with that of rabbit CYP2G1, which
is high spin (Ding and Coon, 1988).
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Immunoblot analysis of CYP2A5 and CYP2G1 in mouse OM
microsomes.
CYP2A5 and CYP2G1 can both be detected on immunoblots
by antibodies to CYP2A10/11 or to rabbit CYP2G1 (Hua et al.,
1997; Ding X, unpublished observations); highly specific antibodies
that can distinguish the two isoforms are not currently available for a
precise determination of the level of each isoforms in OM microsomes. A
polyclonal rabbit antibody against purified, heterologously expressed
CYP2A5 was obtained in this study. However, as shown in figure
2, although this antibody (termed
anti-CYP2A5) detected only a single band in OM microsomes (lane 1), it
reacted with both CYP2A5 and CYP2G1 on immunoblots (lanes 2 and 3).
Further quantitative analysis indicated that anti-CYP2A5 produces
approximately equal band intensities with equal amounts of the two
isoforms applied; thus, it can be used to estimate the combined level
of the two proteins on immunoblots. In experiments not presented, a
quantitative immunoblot analysis with purified CYP2A5 or CYP2G1 as a
standard indicated that the combined level of the two isoforms in mouse
OM microsomes is 
150 pmol/mg protein, or >35% of the total
microsomal P450 determined spectrally.
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Metabolic activation of DCBN.
The activities of purified
CYP2A5 and CYP2G1 in metabolizing DCBN, an OM-specific toxicant, to
DCBN-protein adducts were examined and compared with those of mouse
liver and OM microsomes. As shown in table
1, OM microsomes were 20 times more
active (per mg of microsomal protein) than liver microsomes in the
formation of DCBN-protein adducts, with 33 to 82 times higher turnover
numbers (per nmol P450) at 30 and 3 µM DCBN, respectively. The
activities of the purified, reconstituted, cytochromes were initially
examined with BSA added as a donor of sulfhydryl groups. With BSA
present, both CYP2A5 and CYP2G1 were active toward DCBN, with turnover numbers similar at 3 µM substrate and higher for CYP2A5 at 30 µM
substrate but 20 times lower than that of OM microsomes. However, the
addition of boiled mouse nasal or hepatic microsomes led to a
substantial increase in the turnover numbers for both isoforms, whereas
the addition of purified cytochrome b5 led
to further increases in the activities of CYP2A5 and CYP2G1 to about
one half (for CYP2A5) and one third (for CYP2G1) of that found in OM
microsomes and much higher than activities seen in liver microsomes. Activity was not detected in control reactions containing boiled microsomes alone, with purified P450s omitted (not shown). These data
indicate that both isoforms are active toward DCBN and that the
dramatic difference in DCBN metabolic activities in liver and OM
microsomes is not due to differences in the level of protein targets
for adduct formation.
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Metabolic activation of AP.
Purified CYP2A5 and CYP2G1 were
found to be active in metabolizing AP to 3-OH-AP and, in the presence
of GSH, to GS-AP in a preliminary study (Genter et al.,
1998). The activities of purified CYP2A5 and CYP2G1 in metabolizing AP
to AP-protein adducts as well as 3-OH-AP and GS-AP were further
examined here and compared with those of mouse liver and OM microsomes.
The HPLC profiles of AP metabolites produced by CYP2A5 in a
reconstituted system are shown in figure
3. The only soluble metabolite detected
in the absence of GSH with either purified P450s or in liver and OM
microsomal reactions (not shown) was 3-OH-AP. With the addition of GSH,
GS-AP was detected as the predominant product in addition to 3-OH-AP in
all reactions. Three additional, minor, peaks, which may represent
unidentified products, were also detected (fig. 3D, with retention
times of 4.0, 6.0 and 11.5 min, respectively); the small peak at
~11.5 min was also detected when 3H-GSH was
used with unlabeled AP as substrate (data not shown), indicating that
it may be a degradation product of GS-AP.
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Role of CYP2A5 and CYP2G1 in OM microsomal metabolism of DCBN and
AP.
The role of CYP2A5 and CYP2G1 in microsomal metabolism of DCBN
and AP was examined with the rabbit anti-CYP2A5 antibody used in
immunoblot quantitation. In experiments not shown, anti-CYP2A5 IgG
completely inhibited coumarin hydroxylase activity of both CYP2A5 and
CYP2G1 in a reconstituted system when added at 5 mg IgG/nmol P450.
Thus, the effects of various amounts of anti-CYP2A5 IgG on microsomal
metabolism of AP to AP-protein adducts and of DCBN to DCBN-protein
adducts were examined, with preimmune rabbit IgG added to maintain a
constant concentration of IgG in all reactions. As shown in figure
4, a concentration-dependent inhibition
of OM microsomal activities was observed with both substrates, with maximal inhibitions of 80% of control activities achieved at 3 mg
IgG/mg microsomal protein, confirming that one or both of these
isoforms play a major role in AP and DCBN metabolism in OM microsomes.
The addition of preimmune IgG alone did not cause any inhibition of the
activities (not shown).
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),
and 8-methoxsalen has been found to inhibit heterologously expressed rat CYP2A3 (Liu et al., 1996). As shown in figure
5, A and B, the compounds inhibited the
activity of the two isoforms as well as that of OM microsomes to a
similar extent, suggesting importance of one or both of these isoforms
in metabolic activation of DCBN. However, 8-methoxsalen is a more
potent inhibitor than 5-methoxsalen; with substrate present at 3 µM,
a 50% inhibition was achieved with ~2 µM 8-methoxsalen or ~10
µM 5-methoxsalen. Similar results were obtained with metyrapone (fig.
5C), a known inhibitor of DCBN toxicity in vivo (Brandt
et al., 1990; Walters et al., 1993).
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-naphthoflavone, 4-methylpyrazole,
1-aminobenzotriazole and coumarin, were found to inhibit the activities
of CYP2A5 and CYP2G1 to similar degrees and thus could not be used to
determine the relative roles of the two isoforms in OM microsomal
metabolism.
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Role of CYP1A2 and CYP2E1 in OM microsomal DCBN and AP
metabolism.
The possible role of CYP1A2 in OM microsomal
metabolism of DCBN and AP were examined with Cyp1a2(/)
mice and wild-type littermates. As shown in table
3, rates of formation of AP- and
DCBN-protein adducts were not decreased in OM S9 fractions from
Cyp1a2(/) mice compared with Cyp1a2(+/+)
mice, indicating that CYP1A2 does not play an important role in the
metabolic activation of these compounds in the olfactory mucosa. For
reasons not understood, S9 fractions from the CYP1A2-knockout mice had
significantly higher activity toward DCBN than the wild-type
littermates, although no difference was found with AP as a substrate.
|
Kinetics of AP and DCBN metabolism by purified CYP2A5 and CYP2G1. To evaluate the relative importance of CYP2A5 and CYP2G1 in OM microsomal metabolism of AP and DCBN, we determined the kinetics of AP and DCBN metabolism in a reconstituted system. As shown in table 4, CYP2A5 had only slightly lower Km values than CYP2G1 did toward AP but much higher Vmax values in the formation of both 3-OH-AP and GS-AP. With DCBN, however, CYP2G1 had a much lower Km and lower Vmax values than CYP2A5 did in the formation of GS-DCBN. Thus, it appears that CYP2A5 may play a greater role in OM microsomal metabolism of AP, whereas the relative roles of CYP2A5 and CYP2G1 in DCBN metabolism may be dose dependent, with CYP2G1 playing a more important role at relative low substrate concentrations.
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Discussion |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Metabolic activation of DCBN is believed to go through 2,3- and
3,4-epoxy-DCBN, which form adducts with GSH or protein sulfhydryls (Ding et al., 1996). Several P450 isoforms are known to be
active toward DCBN, including rat CYP2A3, rabbit CYP2A10/11, rabbit
CYP2E1, human CYP2A6 and human CYP2E1 (Ding et al., 1996;
Liu et al., 1996). AP is metabolized either directly or
through yet unidentified intermediates to 3-OH-AP and
N-acetyl-p-benzoquinone imine; the latter is believed to be
the toxic intermediate that reacts with GSH or protein sulfhydryls to
form covalent adducts (Hinson et al., 1980; Dahlin and
Nelson, 1982; Harvison et al., 1988). Several P450s are
known to catalyze this reaction, including human CYP1A2, CYP2E1 and
CYP3A4 and their orthologs in other species (Morgan et al.,
1983; Harvison et al., 1988; Patten et al., 1993;
Lee et al., 1996; Kostrubsky et al., 1997; Zhou
et al., 1997). However, definitive evidence for the P450
isoforms responsible for the metabolic activation of the two compounds
in the olfactory mucosa has not been obtained in previous studies.
OM microsomes are much more active than hepatic microsomes in the
metabolic activation of DCBN and AP, which may contribute to the
tissue-selective toxicity of these compounds. Several lines of evidence
were obtained in the present study that indicate that CYP2A5 and CYP2G1
play major roles in OM microsomal metabolic activation of DCBN and AP.
First, purified CYP2A5 and CYP2G1 demonstrated activity toward the two
toxicants in reconstituted systems. To this end, evidence was also
obtained that CYP2A5 and/or CYP2G1 are abundant P450 isoforms in OM
microsomes; the combined level of the two may account for >35% of
total P450 in this tissue. Second, 5- or 8-methoxsalen provided
parallel inhibition of AP or DCBN metabolic activation by the purified
isoforms and OM microsomal preparations; similar results were obtained
using an inhibitory anti-CYP2A5 antibody that also inhibits CYP2G1. In
addition, the roles of two other P450 isoforms, CYP1A2 and CYP2E1, in
the OM bioactivation of AP and DCBN were ruled out. There was no
increase in the OM microsomal activities in acetone-treated mice
compared with untreated mice, although OM microsomal CYP2E1 levels were 3-fold higher in acetone-treated mice. On the other hand, there was no
decrease in the OM microsomal activities in Cyp1a2(/) mice compared with Cyp1a2(+/+) littermates. These results
are consistent with a previous report suggesting that CYP2E1 may not play a major role in DCBN activation in rat OM microsomes (Eriksson and
Brittebo, 1995) and with a recent study using Cyp1a2(/) mice indicating that CYP1A2 does not play a major role in the OM
toxicity of AP (Genter et al., in press).
The effects of adding boiled liver or nasal microsomes on rates of DCBN- and AP-protein adduct formation in reactions with purified P450s are interesting. With DCBN, the addition of boiled microsomes instead of BSA as a donor of sulfhydryl groups led to a big increase in the rate of adduct formation. Similar increases were observed regardless of whether liver microsomes or nasal microsomes were used, indicating that the differences in liver and OM microsomal activities were not due to potential differences in microsomal proteins that can conjugate the reactive intermediates formed by P450 reaction. With AP, however, no increases were observed with addition of boiled microsomes. It remains to be determined whether the differential stimulation reflects different reactivity of the reactive intermediates formed from the two substrates.
The amino acid sequences of CYP2A5 and CYP2G1 are <60% identical (Hua
et al., 1997). However, the two isoforms appear to be highly
similar in immunological properties, substrate specificity and
inhibitor selectivity. The two proteins are not resolved on immunoblots, and both react with several available antibodies prepared
against CYP2A or CYP2G, including a monoclonal anti-CYP2A6 antibody
from Gentest (data not shown). Both CYP2A5 and CYP2G1 are active toward
a number of substrates, including coumarin, testosterone, progesterone,
4-NP, DCBN and AP, and both are inhibited similarly by all compounds
tested to date. Furthermore, neither isoform appears to be inducible in
the olfactory mucosa by chemical treatments. Although differences in
kinetics parameters for DCBN and AP metabolism were found between
purified CYP2A5 and CYP2G1, it is difficult at present to determine
conclusively their individual roles in metabolic activation in OM
microsomes. Attempts are being made to obtain isoform-specific
antibodies to facilitate in vitro studies, and efforts to
develop knockout mice models lacking either gene are warranted. The
latter animal models will be very useful not only for resolving the
role of the two P450 isoforms in microsomal metabolism but also for
elucidating the role of these tissue-selective or -specific P450 forms
in OM-specific toxicity of numerous foreign compounds, which is
essential for reliable extrapolation of data from animal studies to
risk assessment in humans.
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Acknowledgments |
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We are grateful to Dr. Laurence Kaminsky for providing purified rat CYP1A1 and reading the manuscript. The authors gratefully acknowledge the use of Wadsworth Center Biochemistry and Tissue Culture core facilities.
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Footnotes |
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Accepted for publication February 24, 1998.
Received for publication November 25, 1997.
1 This study was supported in part by National Institutes of Health Grants ES07462 (X.D.), AG13837 (M.B.G.), ES06321 (D.W.N.) and P30-ES06090 (D.W.N.).
Send reprint requests to: Dr. Xinxin Ding, Laboratory of Human Toxicology and Molecular Epidemiology, Division of Environmental Disease Prevention, Wadsworth Center, New York State Department of Health, Empire State Plaza, Box 509, Albany, NY 12201-0509. E-mail: xding{at}wadsworth.org
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Abbreviations |
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P450, cytochrome P450; b5, cytochrome b5; OM, olfactory mucosal; SF9, Spodoptera frugiperta; DCBN, 2,6-dichlorobenzonitrile; BSA, bovine serum albumin; IgG, immunoglobulin G; 4-NP, p-nitrophenol; AP, acetaminophen; 3-OH-AP, 3-hydroxyacetaminophen; GSH, glutathione; GS-AP, 3-glutathion-S-ylacetaminophen; GS-DCBN, glutathione conjugate of DCBN epoxide; HPLC, high performance liquid chromatography.
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References |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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,'-iminodipropionitrile in the rat.
J Pharmacol Exp Ther
263:
1432-1439/) mouse.
Biochem Pharmacol In press./) null mutant mice develop normally but show deficient drug metabolism.
Proc Natl Acad Sci USA
93:
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