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Vol. 290, Issue 1, 281-288, July 1999
Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis (M.A.S., A.R.B.); Department of Entomology, School of Agricultural and Environmental Sciences, University of California, Davis (P.V.C.); and Department of Epidemiology and Preventive Medicine, School of Medicine, University of California, Davis (A.R.B.)
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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Despite their substantially lower levels relative to hepatic
tissue, pulmonary cytochrome P-450 (CYP) monooxygenases play an
important role in the metabolic activation of substrates that cause
lung injury. The target- and species-selective toxicity of a number of
pulmonary toxicants has been attributed to the presence and
distribution of activating enzymes with high
kcat in target airways of susceptible
species. However, experimental demonstration of these concepts and
quantitative assessment of the contribution of individual CYP isoforms
is lacking. This study was undertaken to characterize the catalytic
activities of CYP2F2 with naphthalene, a murine Clara cell toxicant, as
well as with other xenobiotics that either undergo metabolic activation
to cytotoxic intermediates or that function as "isoform-selective" substrates. Recombinant CYP2F2 was produced using the baculovirus expression vector system in Spodoptera frugiperda and
Trichoplusia ni cells, accounting up to ~20% of the
total cellular protein. Incubations containing naphthalene, recombinant
CYP2F2, NADPH-cytochrome P-450 oxidoreductase, and
NADPH-regenerating system metabolized naphthalene with a high
degree of stereoselectivity to
1R,2S-naphthalene oxide (66:1
enantiomeric ratio). The Km and
kcat values, along with the specificity
constant, for naphthalene metabolism by recombinant CYP2F2 were 3 µM, 104 min
1, and 5.8 × 105
M1 s1, respectively.
Recombinant CYP2F2 also metabolized ethoxyresorufin, pentoxyresorufin,
p-nitrophenol, and 1-nitronaphthalene at easily detectable levels. The results from this work suggest that CYP2F2 1)
plays a key role in the species- and cell-selective toxicity of
naphthalene and 2) efficiently metabolizes a number of other substrates, including the lung toxicant 1-nitronaphthalene.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Numerous
studies with laboratory animals have demonstrated the importance of
respiratory epithelial cells as targets for both inhaled and ingested
chemicals. Specifically, nonciliated bronchiolar epithelial (Clara)
cells are very sensitive to toxicants, and this appears to be due to
the metabolic capabilities of the Clara cell (Plopper, 1993
; Gram,
1997). With several lung toxicants such as trichloroethylene,
nitronaphthalene, 3-methylindole, and naphthalene (see Gram, 1997,
Yost, 1997; for review), metabolism by cytochrome P-450 (CYP)
monooxygenases is an obligate step in the formation of toxicologically
active derivatives. The eventual outcome from exposure of a particular
tissue/cell appears to depend on a complex interplay among exposure
levels, the rate of metabolism of both parent compound and reactive
metabolite, and the sensitivity of the cell to the biologically active
derivative. A total of seven CYP monooxygenases have been reported in
the lung, including CYP1A1, CYP1B1, CYP2B, CYP2E1, CYP2F, CYP3A, and
CYP4B1/2. The catalytic activities of a few of these isoforms have been
reported using either purified proteins or recombinant enzymes
(Buckpitt and Cruikshank, 1997, and Yost, 1997, for reviews; Willey et
al., 1996). With the exception of 4-ipomeanol, where the quantitative contributions of CYP2B4 and CYP4B1 in the rabbit were assigned a number
of years ago (Wolf et al., 1982; Domin et al., 1986), the roles of each
of the pulmonary isoforms in substrate turnover have not been assessed fully.
Substantial species differences have been observed in sensitivity to a
variety of lung toxicants. For example, parenteral administration of
naphthalene, 2-methylnaphthalene, dichloroethylene, and
trichloroethylene results in Clara cell necrosis in mice but not in
rats (Plopper, 1993). One of the possible explanations for these
species differences is that there is a pulmonary CYP isoform in the
mouse that shows high catalytic activity with a number of these agents.
The mouse is a particularly sensitive species for naphthalene toxicity,
with the lung being the primary target (Plopper et al., 1992). Previous
work has demonstrated a correlation between the presence of CYP2F2 in
mouse Clara cells with the stereoselective epoxidation of naphthalene
and the site-selective cytotoxicity of this chemical in murine Clara
cells (Buckpitt et al., 1995). A cDNA copy of CYP2F2 mRNA was isolated
from mouse liver and the presence of this mRNA was shown in mouse lung
by Northern blot analysis (Ritter et al., 1991). Immunohistochemical localization with antibodies against purified CYP2F2 also confirmed its
presence in mouse lung Clara cells (Buckpitt et al., 1995). Furthermore, an inhibitory anti-CYP2F2 antibody markedly alters the
ratio of epoxide enantiomers from 10:1 to 1:1 in mouse lung microsomes
(Nagata et al., 1990). CYP2F2 has been expressed at low levels in the
yeast expression system using Saccharomyces cerevisiae and
has been shown to metabolize naphthalene with a high degree of
stereoselectivity (Ritter et al., 1991). This evidence localizes CYP2F2
to the mouse lung Clara cell and strongly suggests its involvement in
the metabolism and, possibly, the toxicity of naphthalene in mouse
lung. Although the yeast-expressed CYP2F2 has been partially
characterized with naphthalene, the system has never been optimized and
kinetic parameters have not been determined. Accordingly, the objective
of this work was to express CYP2F2 at high levels in insect cells and,
using optimized conditions, determine the catalytic activity of the
recombinant protein with both naphthalene and other relevant substrates.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Reagents.
The CYP2F2 cDNA clone in pBluescript
SK was a gift from Dr. J. Ritter (Virginia
Commonwealth University, Richmond, VA). Spodoptera frugiperda (Sf9) and Trichoplusia ni (Tn5) cells were
obtained from American Type Culture Collection (Manassas, VA) and
Invitrogen (Carlsbad, CA), respectively. Ex-Cell 405 medium was
purchased from JRH Biosciences (Lenexa, KS). Grace's medium was from
Life Technologies (Rockville, MD). Mouse NADPH-cytochrome P-450
oxidoreductase (reductase) was purified from liver using standard
procedures (Strobel and Dignam, 1978). The specific activity of the
purified reductase preparation was 3.6 U/mg protein (1 U = 1 µmol cytochrome c reduced/min). Reductase was quantified
by the standard cytochrome c reduction assay (Guengerich,
1994). Conversions from cytochrome c units to molar
concentrations are based on the assumption that pure reductase reduces
55 µmol cytochrome c/min/mg of the protein under the above
assay conditions. Glutathione-S-transferases (GST) from
mouse liver cytosol were purified by standard procedures using affinity
column chromatography (Simons and Vander Jagt, 1981), and activities
were assessed with 1-chloro-2,4-dinitrobenzene as substrate.
Recombinant human cytochrome b5 was a
generous gift from Dr. M. Shet and Dr. R. Estabrook (University of
Texas, Dallas, TX). Unless otherwise stated, all other reagents were
purchased from commercial vendors and were of reagent/analytical grade.
Construction of Recombinant Baculovirus.
The cDNA encoding
murine CYP2F2 was directionally cloned from pBluescript
SK containing the complete CYP2F2 cDNA
(pBluescript SK/2F2) into the multiple cloning
site of pFastBac1 (Life Technologies, Rockville, MD) using two steps.
1) Polymerase chain reaction (PCR) was used to incorporate a
BamHI site immediately before the translational start codon
of CYP2F2 as well as for introducing an intentional silent mutation
(T
C) at position 288 to incorporate a NotI site. The PCR
product was digested with BamHI/NotI, gel
purified, and ligated into the BamHI/NotI sites
in the multiple cloning site of pFastBac1 using T4 DNA ligase. A
recombinant construct (pFastBac1/2F2-5') containing the 5' 288 bp of
the CYP2F2 open reading frame was isolated and sequenced to ensure that
unwanted PCR errors had not occurred during amplification. 2) The
construct (pFastBac1/2F2-5') was linearized with XbaI,
blunt-ended with T4 DNA polymerase, and digested with NotI,
leaving an available NotI end at position 288 and a blunt
end downstream from the multiple cloning site of pFastBac1. The
remainder of the CYP2F2 open reading frame was prepared from
pBluescript SK/2F2 by digestion with
EaeI (leaving a NotI-compatible end as with the
PCR product) and with ScaI (leaving a blunt end two
nucleotides after the stop codon). This fragment was gel purified and
ligated into the linearized pFastBac1/2F2-5'.
1539 of
pBluescript SK/2F2 was isolated and sequenced
to ensure the integrity and orientation of the construct. This
construct (with a 1478-bp insert) consists of the complete CYP2F2 ORF
(1476 bp) plus two 3' nucleotides of untranslated sequence. The
sequence contains one intentional silent mutation, as mentioned.
Recombinant CYP2F2 Expression. Baculovirus stocks were prepared using the Bac-to-Bac expression system (Life Technologies, Rockville, MD). The cDNA encoding CYP2F2 from pFastBac1/2F2 was site-specifically transposed into the baculovirus genome as described in the manufacturer's protocol. The high-molecular-weight genomic DNA was purified from recombinant clones and used to transfect Sf9 cells for the production of baculovirus stocks from various recombinant plasmid constructs. Viral stocks were amplified and stored at 4°C. All baculovirus stocks generated overexpressed a protein product with the same mobility in SDS-polyacrylamide gel electrophoresis (PAGE), yielding similar CYP spectra, and all were capable of metabolizing naphthalene with similar metabolic profiles. Therefore, one stock was chosen for further characterization. Plaque assays showed the viral titer to be 1.2 × 108 plaque-forming units (pfu)/ml.
Sf9 cells grown in complete Grace's insect cell culture medium and Tn5 cells grown in complete Ex-Cell 405 insect cell culture medium were used as hosts for infection. Infections were performed by incubating monolayers of host cells at a density of 2 × 105 cells/cm2 in 6-well plates or culture flasks with recombinant virus at a multiplicity of infection (m.o.i.) of 1 pfu/cell. Hemin chloride was added 24 h postinfection (p.i.) to a final concentration of 5 µg/ml. Alternatively, 5-aminolevulinic acid (ALA) and ferric citrate (FC) were both added at a concentration of 100 µM at the time of infection to avoid a substantial 422-nm absorption during spectral analysis due to contaminating hemin in the preparations. Recombinant CYP2F2 was harvested at 72 h p.i. Cell lysates were prepared by pelleting cells at 1000g for 10 min followed by washing twice with ice-cold PBS. Pellets were frozen immediately in liquid nitrogen for 5 to 10 min to lyse cells, thawed at 37°C in the appropriate buffer supplemented with 100 µg/ml phenylmethylsulfonyl fluoride, and homogenized in a glass-glass tissue grinder on ice. Buffers consisted of 0.1 M phosphate buffer, pH 7.4, when cell lysates were to be used immediately; 0.1 M phosphate buffer, pH 7.4, with 20% glycerol and 0.1 mM EDTA for storage at
80°C; or 0.1 M phosphate buffer, pH 7.4, with 0.25 M sucrose and 0.1 mM EDTA for microsomal preparations. Cell
lysate preparations were adequate for conducting metabolism
experiments. Microsomes were prepared by differential centrifugation of
cell lysates at 10,000g for 15 min at 4°C followed by
100,000g for 90 min at 4°C. CYP levels were determined by
obtaining difference spectra of sodium dithionite-reduced versus
CO-bubbled samples at 500 to 400 nm according to Omura and Sato (1964).
Noninfected Tn5 or Sf9 cells were used as negative controls and were
supplemented and prepared exactly as infected cells.
SDS-PAGE and Western Blotting.
Proteins from either Tn5 cell
lysates or microsomes were separated by SDS-PAGE using premade 1-mm
10% Tris-glycine gels (Novex, San Diego, CA). Bands were identified by
Coomassie blue staining and by Western blotting. Western blot analysis
was performed using the method of Laemmli (1970). Recombinant CYP2F2
was identified using rabbit anti-CYP2F2 (Nagata et al., 1990). Blots
were blocked (30 min at room temperature) with 3% BSA/1% nonfat dried
milk, hybridized (overnight at 4°C) with rabbit anti-CYP2F2 (1:20,000 dilution); hybridized (15 min at room temperature) with goat
anti-rabbit IgG linker antibody (1:100 dilution), hybridized (15 min at
room temperature) with a rabbit peroxidase anti-peroxidase complex (1:2000 dilution) (Cappel, Organon Teknika, Durham, NC), and stained (5 min at room temp) with 3,3'-diaminobenzidine.
Measurement of Naphthalene Metabolism.
The rates of
naphthalene metabolism were assessed in incubations containing
recombinant CYP2F2 (quantity determined spectrally), reductase
(quantity determined by cytochrome c activity),
NADPH-regenerating system (0.25 U of glucose-6-phosphate dehydrogenase,
14 mM glucose-6-phosphate, 2.18 mM NADP, and 1 mM
MgCl2), 1 mM glutathione (GSH), and 2.5 U GST in
a total volume of 250 µl (0.1 M
Na2HPO4, pH 7.4). CYP2F2 and reductase concentrations are specified in the figure legends. GSH
and GST were included for the trapping of reactive epoxides as stable
GSH conjugates (Fig. 1). Incubations were
performed in a shaking water bath at 37°C for the times specified in
the figure legends. Reactions were quenched on ice by the addition of
two volumes of methanol. Protein was removed by centrifugation, and the
supernatants were evaporated under reduced pressure. GSH conjugates
were separated by reversed phase HPLC and were quantified by peak areas
using a HP1100 UV detector at 260 nm (Buckpitt et al., 1987). A
variation to this method was a change to a new mobile phase system
(0.06% triethylamine, pH 3.1/acetonitrile) with a gradient of
acetonitrile ranging from 5% to 7% over the first 60 min of a 100-min
run. GSH conjugate standards were prepared by synthesis from
naphthalene oxide and GSH and were purified by preparative HPLC.
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Measurement of Ethoxyresorufin, Pentoxyresorufin, and
p-Nitrophenol Metabolism.
Incubations with
p-nitrophenol, ethoxyresorufin, and pentoxyresorufin were
carried out in 250-µl volumes (0.1 M
Na2HPO4, pH 7.4) containing
5 pmol of recombinant CYP2F2, 8.5 pmol of mouse liver reductase, and
NADPH-regenerating system. Incubations were performed in a shaking
water bath at 37°C for 20 min. Ethoxyresorufin and pentoxyresorufin
reactions were quenched on ice with two volumes of methanol. Insoluble
material was removed by centrifugation, and the supernatants were
diluted with an equal volume of water for HPLC analysis. Resorufin was
analyzed by reversed phase HPLC with fluorescence detection as
described previously (
ex = 535 nm;
em = 585 nm) (Plopper et al., 1993).
.
Measurement of 1-Nitronaphthalene Metabolism.
Incubations
with 1-nitronaphthalene were carried out in 250-µl volumes containing
recombinant CYP2F2, reductase, and NADPH-regeneration system as
described above. Incubations were performed in the presence of
3H-GSH (0.25 mM, 6500 dpm/nmol) and GST (2.5 U)
for the trapping of any reactive epoxides as GSH conjugates.
Incubations were performed in a shaking water bath at 37°C for 30 min. Reactions were stopped, and samples prepared for analysis as with
naphthalene (see above). GST conjugates were separated by reversed
phase HPLC using a variation of a method by Watt et al. (1998). The
change in the method was to a 0.06% triethylamine, pH 3.1/acetonitrile
mobile phase system, with an acetonitrile gradient ranging from 5% to
16% over the first 60 min of a 110-min run. The mobile phase flow rate
was 1 ml/min (K. C. Watt, personal communication). Column effluent was monitored at 256 nm using a HP1100 UV detector. Fractions were
collected at 0.5-min intervals and counted using a Beckman LS5000TD
scintillation counter.
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Baculovirus Expression of Recombinant CYP2F2.
The expression
of recombinant CYP2F2 was assessed by measuring CO-difference spectra,
by SDS-PAGE with Coomassie blue staining, by Western blotting, and, in
some instances, by catalytic activity with naphthalene as substrate.
Experimental conditions, including the host cell line (Sf9 and Tn5
cells), m.o.i., harvest time after infection, and the use of various
supplements, were varied to optimize the generation of functional
recombinant protein. Tn5 cells infected with recombinant baculovirus
and incubated for 72 h in medium supplemented with ALA/FC produced
easily detectable quantities of CYP with a Soret maximum at 451 nm
(Fig. 2). The levels of CYP measured by
difference spectra were higher in Tn5 cells than in Sf9 cells despite
the finding that similar quantities of the 50-kDa protein were present
(determined by Coomassie staining; data not shown). Spectra could be
obtained by supplementing Tn5 cells with a heme source (hemin chloride
or ALA/FC), although the addition of hemin chloride yielded a
substantial 422-nm absorption that was also present in negative
controls. Supplementation with ALA/FC gave very little or no 422-nm
absorption but resulted in lower overall levels of CYP.
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). The 1R,2S-naphthalene oxide
(conjugate 2) was produced at a 66:1 enantiomeric ratio over the
1S,2R-naphthalene oxide (conjugates 1 and 3). No
naphthalene metabolites were obtained from incubations containing
noninfected cell lysates under similar conditions. Reactions containing
recombinant CYP2F2 were inhibited by either bubbling carbon monoxide
before incubations or deleting the NADPH-regenerating system (data not shown).
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Optimization of Naphthalene Metabolism.
Before conducting
kinetic studies, optimal incubation conditions for naphthalene
metabolism by recombinant CYP2F2 were established. Naphthalene
metabolism was not detected in incubations containing noninfected Tn5
cell lysates, CYP reductase, NADPH-regenerating system, and
naphthalene. Similar incubations containing CYP2F2 but without
supplemental reductase revealed very low rates of naphthalene
metabolism (corresponding to ~3% of total naphthalene metabolism in
complete incubations), indicating a small amount of endogenous electron
donor for CYP2F2. Increasing quantities of CYP reductase (assayed by
cytochrome c reduction) were added to incubations containing
2.5 pmol of CYP2F2 and other components necessary to trap naphthalene
epoxides as GSH conjugates. As the ratio (nmol/nmol) of CYP reductase
to CYP was increased from 0.1:1 to 1.4:1, the rates of naphthalene
metabolism increased from less than 2 nmol/min/nmol CYP2F2 to more than
90 nmol/min/nmol CYP2F2 (Fig. 6). No
further increases were noted with ratios above 1.4:1, and, accordingly,
a ratio of 1.7:1 was used for all further metabolism experiments. No
alterations in the ratio of 1R,2S- to
1S,2R-epoxide were noted at the varying
quantities of reductase in the incubation. Preincubation of the
reductase with CYP2F2 was not necessary to obtain optimal catalytic
activities (data not shown).
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Kinetics of Naphthalene Metabolism by CYP2F2.
Before kinetic
studies, linearity was established with protein and with time, and all
kinetic studies were conducted in the linear portion of the curves. The
kinetic parameters (Km and
kcat) for the metabolism of
naphthalene by recombinant CYP2F2 were established with this optimized
system. A preliminary experiment indicated a very low apparent
Km; accordingly, incubation times were
shortened to 4 min to decrease the percentage of naphthalene consumed
in the reaction. Substrate concentrations varied from 0.0025 to 1.0 mM
naphthalene. Data were analyzed by nonlinear regression (Fig. 7) for the estimation of kinetic
parameters. The Km of CYP2F2 with
naphthalene was 3 µM with a kcat of
104 min1. The specificity constant of CYP2F2
for naphthalene was calculated to be 5.8 × 105
M1
s1. No alterations in the ratio of
1R,2S- to 1S,2R-epoxide
were noted at the varying concentrations of naphthalene in the
incubation.
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Metabolism of Other Substrates by CYP2F2. Ethoxyresorufin, pentoxyresorufin, and p-nitrophenol are thought to be isoform-selective substrates for other CYPs in the lung (CYP1A1, CYP2B, and CYP2E, respectively). Incubations containing recombinant CYP2F2, reductase, and the necessary cofactors metabolize all three substrates at easily detectable rates under similar conditions as with naphthalene. Comparing the rates of metabolism for these substrates with published activities for their respective isoforms indicated that the turnover of both ethoxyresorufin and pentoxyresorufin by recombinant CYP2F2 is dramatically slower than that for CYP1A1/1B1 or CYP2B, respectively (Table 2). In contrast, metabolism of p-nitrophenol by CYP2F2 and CYP2E1 occurs at very similar rates.
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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A number of pulmonary toxicants, including naphthalene, show
highly species-selective toxicity. Naphthalene shows differential toxicity to the mouse and the rat. The i.p. administration of naphthalene at doses as low as 50 mg/kg produces detectable Clara cell
necrosis in mice; toxicity is not discernible in rat lungs even at
doses of 1600 mg/kg (Plopper et al., 1992). These species differences
in susceptibility correlate well with the rates of naphthalene
metabolism to the epoxides (see Fig. 1) in both lung microsomes
(Buckpitt et al., 1992) and dissected airway explants (Buckpitt et al.,
1995). Furthermore, there are no striking differences in either epoxide
hydrolase- or GST-mediated detoxification pathways for naphthalene
oxide, which would account for the unusual susceptibility of the mouse
compared with the rat (Lorenz et al., 1984). These factors suggest that
the initial metabolic activation of naphthalene is a key event in
determining the species susceptibility to this compound. The data
presented here, demonstrating very high turnover numbers with
naphthalene as substrate (kcat 
104 min1) and a biologically relevant
Km (3 µM), support the importance of
CYP2F2 in determining species-selective toxicity of naphthalene.
A number of CYP isoforms have been expressed at high levels using the
baculovirus-insect cell system, including CYP2A6 (Chen et al., 1997),
CYP2D6 (Paine et al., 1996), CYP2E1 (Patten and Koch, 1995), and CYP3A4
(Buters et al., 1994). The advantages of the system, namely that of
high levels of expression and targeting to the membrane fraction in
comparison with other heterologous expression systems, have been
described in detail by others (Lee et al., 1996). In this work, the
levels of CYP2F2 in insect cell lysates (2.7 nmol/mg protein) are
higher than those reported for CYP2D6 (0.1-0.2 nmol/mg cell lysate),
CYP2E1 (0.5-0.8 nmol/mg cell lysate), and CYP3A4 (0.4 nmol/mg cell
lysate). The production of recombinant CYP2F2 was similar in yield to
that of the polyhedrin protein of wild-type AcNPV-infected cells. The
time of optimal expression was similar to that of studies reported
previously with other CYP monooxygenases at ~72 h p.i. These data
confirm the efficient expression of recombinant CYP2F2 in the
baculovirus expression system. The data showing cross-reactivity with
anti-CYP2F2 antibody confirm that recombinant CYP2F2 possesses the
antigenic properties of the native pulmonary enzyme. Furthermore, the
metabolism data confirm that recombinant CYP2F2 possesses the
functional characteristics of the native enzyme-metabolizing
naphthalene to the 1R,2S-oxide over the
1S,2R-oxide at a ratio of 66:1, which is similar
to the ratio of 30:1 in mouse lung microsomes (Buckpitt et al., 1992).
The results of the current study, demonstrating the requirement
for the addition of saturable levels of CYP reductase to achieve optimal catalytic activity, was similar to other work with
baculovirus-expressed CYPs such as CYP3A4 (Buters et al., 1994), CYP2D6
(Paine et al., 1996), and CYP2E1 (Patten and Koch, 1995). We are
confident, having reached saturation with reductase, that maximum
turnover rates of naphthalene were achieved with recombinant CYP2F2.
Similar experiments in which mouse lung microsomes were supplemented
with additional purified reductase yielded no enhancement of
naphthalene turnover (data not shown), implying that CYP2F2 operates
under saturating reductase conditions in microsomal preparations.
In subsequent metabolism experiments using CYP2F2 with different reductase preparations, we have found that the addition of 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate is necessary to reach the optimum turnover rates with naphthalene as reported here. The addition of 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate, however, does not stimulate metabolism beyond the catalytic activities presented here.
Earlier work has demonstrated that the addition of cytochrome
b5 can markedly increase the catalytic
activities of some CYP isoforms, including CYP2E1 (Patten and Koch,
1995) and CYP2F3 (Wang et al., 1998). There was a slight increase in
the metabolism of naphthalene by CYP2F2 when supplemented with
cytochrome b5. Although the
differences were statistically significant (p < .05), metabolism of naphthalene increased only marginally by 18% to 20% at
a 1:1 or 3:1 ratio (nmol cytochrome
b5/nmol CYP). In comparison, the
addition of cytochrome b5 at similar
ratios with CYP2E1 incubations increased the metabolism of
N-nitrosodimethylamine, ethoxycoumarin, and
p-nitrophenol by 3-, 6-, and more than 10-fold,
respectively. The addition of cytochrome
b5 at similar ratios with CYP2F3
incubations increased the metabolism of 3-methylindole by 6-fold. In
relation to these examples, supplementation of CYP2F2 with cytochrome
b5 did not increase the metabolism of
naphthalene substantially.
Comparative data with CYPs isolated from different species must
be interpreted cautiously; slight sequence variations between species
may markedly influence the catalytic activities of the proteins.
Ethoxyresorufin, pentoxyresorufin, and p-nitrophenol are
used as isoform-selective substrates for CYP1A1, CYP2B, and CYP2E1,
respectively. The current work shows that recombinant CYP2F2
metabolizes both ethoxyresorufin and pentoxyresorufin at rates that are
considerably slower than the turnover observed with
ethoxyresorufin-O-dealkylation by CYP1A1 (37 nmol/min/nmol CYP; Buters et al., 1995) and with
pentoxyresorufin-O-dealkylation by CYP2B (1.18 nmol/min/nmol
CYP2B4 or 3.15 nmol/min/nmol CYP2B5; Szklarz et al., 1996). These data
indicate that the presence of CYP2F2 would likely contribute very
little when metabolism assays are performed with these diagnostic
substrates. In contrast, even under unoptimized incubation conditions,
recombinant CYP2F2 metabolizes p-nitrophenol at a rate (2.5 nmol/min/nmol CYP) that is close to the rate reported with recombinant
CYP2E1 under similar conditions (2.01 and 4.00 nmol/min/nmol for human
and rat CYP2E1, respectively; Chen et al., 1996). These data indicate
the need to evaluate results using "isoform-specific" substrates,
at least in the case of CYP2E1, with more caution.
Recombinant CYP2F2 also metabolizes the lung toxicant 1-nitronaphthalene to metabolites capable of reacting with GSH (as with naphthalene metabolites). The rate of formation of these reactive species totaled 1.03 ± 0.27 nmol/min/nmol CYP, twice the turnover obtained from similar incubations containing mouse liver microsomes and similar to the turnover by mouse lung microsomes (1 nmol/min/nmol CYP; K. C. Watt, personal communication). These data provide preliminary evidence that CYP2F2 catalyzes the conversion of 1-nitronaphthalene to reactive metabolites at a rate similar to, if not higher than, that observed in mouse lung or liver tissues.
A total of four isoforms from the CYP2F family have been cloned, and
limited studies of catalytic activity have been reported. CYP2F1, from
human lung, has been expressed using vaccinia virus-infected HepG2
cells (Nhamburo et al., 1990). Recombinant CYP2F1 catalyzed the
dealkylation of ethoxycoumarin, propoxycoumarin, pentoxyresorufin, and
benzyloxyresorufin (Nhamburo et al., 1990) and the metabolic activation
of 3-methylindole (Thornton-Manning et al., 1996). CYP2F3, cloned from
goat lung and expressed in Escherichia coli (Wang et al.,
1998), metabolizes 3-methylindole to reactive metabolites (trapped as
mercapturic acid conjugates) with an apparent
Km value of 0.34 mM and a
Vmax value of 0.55 nmol/min/nmol CYP.
These kinetic data along with antibody inhibition studies that indicate
~85% of the microsomal metabolism of 3-methylindole is catalyzed by CYP4B2 suggest that CYP4B2 is probably responsible for the metabolic activation of the substrate, at least in the goat. The high turnover rates observed in the current work (104 min1)
along with the low Km value (3 µM)
supports the view that CYP2F2 is important in the metabolic activation
of naphthalene in vivo.
A fourth member of the CYP2F family has been cloned from rat,
sequenced, and expressed in Tn5 cells (CYP2F4; Baldwin et al., 1998).
The deduced amino acid sequence is 93% and 80% identical with the
mouse and human, respectively. Current studies that focus on defining
the kinetics and stereochemistry of naphthalene metabolism by
recombinant CYP2F4 will help determine whether the lack of sensitivity
of rat lung to naphthalene is due to low CYP2F levels or to low
catalytic activities of this protein.
Whether a particular isoform of CYP is responsible for the metabolic
activation of a lung toxicant is dependent not only on the kinetics of
metabolism of that toxicant but also on the abundance of the protein
within the target cells. Using the techniques developed by Plopper et
al. (1991) for preparing subcompartments of the lung, we mapped out the
total CYP levels in airways of the mouse. Using the recombinant protein
as a standard, we are currently determining the levels of CYP2F2 in
these individual lung compartments of the mouse by Western blot
analysis. Preliminary data indicate that CYP2F2 is a very abundant
isoform, if not the majority of CYP, in mouse airways.
This study demonstrates high catalytic activity of CYP2F2 toward naphthalene at biologically relevant levels (Km = 3 µM) and provides limited data on the metabolism of the lung toxicant 1-nitronaphthalene. The data generated in this work, together with the relative abundance of CYP2F2 in mouse airways, and the sensitivity of mouse lung to toxicity raise an interesting question in regard to the possible involvement of CYP2F2 in rendering the mouse lung inherently sensitive to a number of metabolically activated compounds. Current work to determine kinetic parameters for CYP2F2 with a variety of metabolically activated lung toxicants (including those mentioned above) should address the possible in vivo importance of CYP2F2 in contributing to what appears to be the unique sensitivity of the mouse to metabolically activated lung toxicants.
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Acknowledgments |
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We thank Dr. Joe Ritter (Virginia Commonwealth University) for providing the CYP2F2 cDNA clone; Dr. John Chandler for his advice in the subcloning of CYP2F2; Dr. Byung-Rae Jin for his assistance and advice in the production of recombinant CYP2F2; Dr. Manju Shet, Dr. Ron Estabrook, and Dr. Alan Conley for providing the cytochrome b5 necessary to conduct our experiments; and Dr. Katherine Watt for providing her method for the analysis of 1-nitronaphthalene metabolism.
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Footnotes |
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Accepted for publication March 16, 1999.
Received for publication November 30, 1998.
1 This work was supported by National Institute of Environmental Health Sciences Grants ES08408, ES04699, and ES04311. University of California, Davis is a Center for Environmental Health Sciences (Grant ES05711), and support for core facilities used in this work is gratefully acknowledged. M.A.S. is supported by NIEHS Predoctoral Fellowship ES05707.
Send reprint requests to: Dr. Michael Shultz, Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, CA 95616. E-mail: mashultz{at}ucdavis.edu
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Abbreviations |
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Sf9, Spodoptera frugiperda; Tn5, Trichoplusia ni; pfu, plaque-forming units; GST, glutathione-S-transferase; AcNPV, Autographa californica nuclear polyhedrosis virus; PAGE, polyacrylamide gel electrophoresis; p.i., postinfection; m.o.i., multiplicity of infection; GSH, glutathione; ALA, 5-aminolevulinic acid; FC, ferric citrate; PCR, polymerase chain reaction.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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