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Vol. 31, Issue 4, 384-391, April 2003
Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, North Carolina
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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Cytochrome P450 (P450) enzymes are major catalysts involved in the
metabolism of xenobiotics and endogenous substrates such as
testosterone (TST). Major TST metabolites formed by human liver microsomes include 6
-hydroxytestosterone (6-OHTST),
2-hydroxytestosterone (2-OHTST), and 15-hydroxytestosterone
(15-OHTST). A screen of 16 cDNA-expressed human P450 isoforms
demonstrated that 94% of all TST metabolites are produced by members
of the CYP3A subfamily with 6-OHTST accounting for 86% of all TST
metabolites. Similar Km values were observed
for production of 6-, 2-, and 15-OHTST with human liver
microsomes (HLM) and CYP3A4. However, Vmax
and CLint were significantly higher for 6-OHTST than
2-OHTST (~18-fold) and 15-OHTST (~40-fold). Preincubation of
HLM with a variety of ligands, including chemicals used in military
deployments, resulted in varying levels of inhibition or activation of
TST metabolism. The greatest inhibition of TST metabolism in HLM was following preincubation with organophosphorus compounds, including chlorpyrifos, phorate, and fonofos, with up to 80% inhibition noticed
for several metabolites including 6-OHTST. Preincubation of CYP3A4
with chlorpyrifos, but not chlorpyrifos-oxon, resulted in 98%
inhibition of TST metabolism. Phorate and fonofos also inhibited the
production of most primary metabolites of CYP3A4. Kinetic analysis
indicated that chlorpyrifos was one of the most potent inhibitors of
major TST metabolites followed by fonofos and phorate. Chlorpyrifos,
fonofos, and phorate inhibited major TST metabolites noncompetitively
and irreversibly. Conversely, preincubation of CYP3A4 with
pyridostigmine bromide increased metabolite levels of 6-OHTST and
2-OHTST. Preincubation of human aromatase (CYP19) with the test
chemicals had no effect on the production of the endogenous estrogen,
17-estradiol.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The
cytochrome P450 (P4501) monooxygenase system is
comprised of a superfamily of heme-containing enzymes, expressed in
many mammalian tissues with the highest levels found in liver, and capable of catalyzing the metabolism of a wide range of both endogenous and exogenous substrates (Nelson et al., 1996
). Human CYP3A4 is one of
the most important and most abundant drug-metabolizing P450
isoforms in human liver microsomes and accounts for approximately 40%
of the total P450 in human liver microsomes (Lehmann et al., 1998).
CYP3A4 not only metabolizes xenobiotics but is also responsible for the
metabolism of endogenous compounds, such as steroid hormones. Human
CYP3A4 plays an important role in the metabolism of testosterone (TST),
androstenedione (AD), and progesterone (Waxman et al., 1988). Direct
and indirect approaches have been employed to show that isoforms
belonging to the CYP3A subfamily are the major contributors to
6-hydroxylation of testosterone as well as the production of several
minor metabolites (Waxman et al., 1988, 1991; Yamazaki and Shimada,
1997).
In the human male, TST is the major circulating androgen. TST is
essential for the development and maintenance of specific reproductive
tissues as well as for other characteristic male properties such as
control of spermatogenesis, retention of nitrogen, promotion of muscle
strength, hair growth, bone density, and many aspects of sexually
dimorphic behavior (Nieschlag and Behre, 1998; Wilson et al., 1998).
Maintaining hormonal balance relies upon a number of variables
including rate of hormone synthesis, interactions among hormones, and
rates of secretion, transport, and metabolism. P450s are a major
controlling element in the maintenance of proper steroid hormone levels
in mammalian systems. Exposure to foreign compounds can exert changes
in endocrine function both directly (hormone agonists or antagonists)
or indirectly (altering circulating levels of hormones by influencing
rates of hormone synthesis or metabolism) that can severely affect
steroid hormone action (Wilson and LeBlanc, 1998). Steroids such as TST
are hydroxylated by P450 in a regioselective and stereoselective manner
(Waxman et al., 1988). It follows that perturbation of the P450 system
by xenobiotics may in turn affect the subsequent metabolism and
disposition of TST. Perturbations in TST metabolism may affect levels
of circulating TST with possible reproductive and other consequences,
including further modulation of the expression of some P450 proteins.
Following the Gulf War some veterans reported illnesses which may have
been the result of chemical exposures. Some studies of these veterans
have concluded that significant correlations between perceived
illnesses and chemical use exist (Haley and Kurt, 1997). The reported
chemical exposures included the insect repellent
N,N-diethyl-m-toluamide (DEET), insecticides such
as permethrin and chlorpyrifos to protect against insect borne diseases and the neuroprotective agent pyridostigmine bromide to protect against
possible nerve gas attack. It has been reported that chlorpyrifos and
DEET are metabolized by human P450s (Tang et al., 2001; Usmani et al.,
2002) and that interactions of Gulf War related chemicals can inhibit
or induce the P450s involved in their metabolism (Usmani et al., 2002).
Other studies have reported that interaction of Gulf War related
chemicals could produce greater than additive toxicity in rats and mice
(Chaney et al., 1997; McCain et al., 1997), increased neurotoxicity in
hens associated with increased inhibition of brain acetylcholinesterase
and Neurotoxicity Target Esterase (Abou-Donia et al., 1996a,b), and
neurobehavioral deficit associated with significant inhibition of
brainstem acetylcholinesterase activities in rats (Abou-Donia et al.,
2001). However, no studies have been carried out to examine the
induction or inhibition potential of these or related compounds on
human P450-mediated metabolism of steroid hormones, such as TST. An
understanding of how Gulf War related chemicals affects the metabolism
of TST could aid in the evaluation of the possible role that these
chemicals may play in deployment-related illnesses.
The main objectives of present study were to identify human liver P450 isoforms responsible for TST metabolism and the products of their activity using an improved HPLC method, to study the effects of various deployment-related chemicals on the metabolism of TST using HLM and CYP3A4, and to study the effects of the test compounds on human aromatase (CYP19).
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Chemicals.
DEET, chlorpyrifos, chlorpyrifos-oxon, phorate, fonofos, deltamethrin,
fipronil, imidacloprid, and permethrin (isomeric mix 78%
trans-20% cis) were purchased from Chem Service
(West Chester, PA). Pyridostigmine bromide was purchased from Roche
Diagnostics (Indianapolis, IN). 6
-, 15-, 15-, 7-, 6-,
16-, 16-, 2-, 2-, 11-OHTST, 11-ketotestosterone
(11-KTST), 11-hydroxyandrostenedione (11-OHAD), AD, and
4-hydroxyandrostenedione (4-OHAD) were purchased from Steraloids
(Newport, RI). HPLC grade water, methanol, acetonitrile, and
tetrahydrofuran were purchased from Fisher Scientific (Pittsburgh, PA).
TST, 17-estradiol, and all other chemicals were purchased, if not
specified, from Sigma-Aldrich (St. Louis, MO).
Human Liver Microsomes and Human P450 Isoforms. Pooled human liver microsomes (HLM) (pooled from 21 donors) and human P450 isoforms expressed in baculovirus infected insect cells (Sf9) (BTI-TN-5B1-4), CYP1A1, 1A2, 2B6, 3A4, 3A5, 3A7, 4A11, 2B6, 2C8, 2A6, 2C9*1 (Arg114), 2C9*2 (Cys144), 2C9*3 (Leu359), 2C18, 2C19, 2D6*1 (Val374), 2E1, and human aromatase (CYP19) were purchased from BD Gentest Corporation.
In Vitro TST Metabolism. Metabolic activity assays for human P450 isoforms were performed by incubation of TST (final concentrations, 250 µM) with an NADPH-regenerating system (0.25 mM NADP, 2.5 mM glucose 6-phosphate, and 2 U/ml glucose-6-phosphate dehydrogenase) in specific buffers recommended by the supplier (BD Gentest Corporation). For CYP1A1, 1A2, 2E1, 2C8, 2D6*1 (Val374), 3A4, 3A5, 3A7, 2B6, 2C18, 2C19, and an insect cell control, a 100 mM potassium phosphate buffer with 3.3 mm MgCl2 (pH 7.4) was used. For 2C9*1 (Arg114), 2C9*2 (Cys144), 2C9*3 (Leu359), 4A11, and 2A6, a 100 mM Tris-HCl buffer with 3.3 mM MgCl2 (pH 7.5) was used. After preincubation at 37°C for 5 min, the reactions were initiated by the addition of ice-cold P450 isoforms (final P450 contents 50 pmol/ml) for 30 min at 37°C. The controls were performed under identical conditions with the insect cell control.
Enzyme kinetic assays for HLM and CYP3A4 were performed by incubation of serial concentrations of TST (final concentrations, 9.375-500 µM) with HLM (final protein concentration, 1 mg/ml) or CYP3A4 (final concentration, 50 pmol/ml) in 100 mM potassium phosphate buffer (pH 7.4 at 37°C) containing 3.3 mM MgCl2. After preincubation at 37°C for 5 min, the reactions were initiated by the addition of ice-cold HLM or CYP3A4 for 10 min. The effects of test chemicals on TST metabolism were examined in HLM and CYP3A4 after preincubation with test compounds. The HLM (final protein concentration, 1 mg/ml) or CYP3A4 (final concentration, 50 pmol/ml) were incubated with individual test compounds (final concentration, 100 µM), NADPH-generating system, and 100 mM potassium phosphate buffer with 3.3 mM MgCl2, pH 7.4, for 5 min at 37°C before adding TST (final concentration, 250 µM). Range finding assays were conducted for chlorpyrifos, fonofos, and phorate inhibition of TST major metabolites. Varying concentrations of chlorpyrifos, fonofos, and phorate (0.5-100 µM) were incubated with CYP3A4 (final concentration, 50 pmol/ml), NADPH-generating system, and 100 mM potassium phosphate buffer with 3.3 mM MgCl2, pH 7.4, for 5 min at 37°C before adding TST (final concentration, 100 µM). Reactions were terminated and analyzed as described above. With selected concentration levels based on the range finding assay, the mode of chlorpyrifos, fonofos, and phorate inhibition on TST major metabolites was investigated. For Michaelis-Menten plots, chlorpyrifos (2 µM), fonofos (5 µM), and phorate (30 µM) were incubated with CYP3A4 (final concentration, 50 pmol/ml), NADPH-generating system, and 100 mM potassium phosphate buffer with 3.3 mM MgCl2, pH 7.4, for 5 min at 37°C before adding TST (final concentration, 9.375-500 µM). To demonstrate whether chlorpyrifos inhibition is reversible or irreversible, incubations with and without chlorpyrifos (2 µM) were conducted with varying concentrations of CYP3A4 (0.78-6.25 pmol), NADPH-generating system, and 100 mM potassium phosphate buffer with 3.3 mM MgCl2, pH 7.4, for 5 min at 37°C before adding TST (final concentration, 100 µM). To determine (inhibition constant) Ki values, chlorpyrifos (1-8 µM), fonofos (1-25 µM), and phorate (10-100 µM) were incubated for 5 min at 37°C with CYP3A4 (final concentration, 50 pmol/ml), NADPH-generating system, and 100 mM potassium phosphate buffer with 3.3 mM MgCl2, pH 7.4, prior to adding TST (final concentrations, 50, 100, or 200 µM). Ki values were calculated from Dixon plots. Since cytochrome b5 (b5) is not coexpressed with CYP3A5 as supplied by BD Biosciences (San Jose, CA), a comparison of CYP3A5 metabolism of TST was made using 10 pmol 3A5 with and without addition of 20 pmol b5. Human aromatase (CYP19) catalyzes the conversion of TST to estradiol. To study the effects of the test chemicals on this conversion, test compounds (final concentration, 200 µM) or a well known competitive inhibitor, 4-OHAD (final concentration, 200 µM) were incubated with CYP19 (final concentration, 50 pmol/ml), NADPH-generating system, and 100 mM potassium phosphate buffer with 3.3 mM MgCl2, pH 7.4, for 5 min at 37°C before adding TST (final concentration, 100 µM). The reaction was terminated after an additional 10 min, and supernatant was analyzed for 17-estradiol
concentration by HPLC.
All assays were conducted in triplicate. All reactions were terminated
by the addition of an equal volume of methanol and vortexing. After
10-min centrifugation at 15,000 rpm in a microcentrifuge, the
supernatants were analyzed for TST metabolite concentrations by HPLC.
The protein concentrations and incubation times used in the assays were
found to be in the linear range in preliminary experiments. No
metabolites were detected when incubations were carried out in the
absence of an NADPH-generating system.
Analysis of Metabolites by HPLC.
Metabolites were analyzed using a Shimadzu HPLC system (Kyoto, Japan).
The Shimadzu HPLC system (Kyoto, Japan) used in this study consisted of
one pump (LC-10AT VP), a four-position solvent selection proportioning
valve (FCV-10AL VP), a degasser (DUG-14A), a Shimadzu autoinjector
(SIL-10AD VP), and a Shimadzu UV/VIS detector (SPD-10AV VP). All system
components were controlled through the Shimadzu powerline firmware.
Data were collected via a Shimadzu system controller (SCL-10A VP) and
analyzed using CLASS-VP 4.3 software. A reverse phase HPLC method was
modified based on the HPLC method of Purdon and Lehman-McKeeman (1997),
for the separation of TST and its potential metabolites. The mobile
phase for pump A was 5% tetrahydrofuran, 95% water, for pump B 100%
methanol. A gradient system was employed in the following manner: 0 to
1 min (30% B), 1 to 10 min (30-60% B), 10 to 22 min (60-65% B), 22 to 28 min (65-80% B), 28 to 30 min (80-90% B), 30 to 32 min (90%
B), 32 to 34 min (90-30% B), and 34 to 36 min (30% B). The flow rate
was 0.5 ml/min. Metabolites were separated by a Prodigy column
[Prodigy 3 µ, 150 × 4.6 mm, ODS (3), 100A; Phenomenex, Rancho
Palos Verdes, CA] and detected at 247 nm. A summary of the retention
times of TST and 14 TST metabolites are presented in Table
1. The limits of detection for most of
TST metabolites were approximately 0.04 µM except for 6-OHTST
(0.15 µM) and 4-OHAD (0.30 µM). Standards of TST metabolites were
made in methanol and 50-µl standard or sample injected on HPLC.
Concentrations of metabolites were obtained by extrapolation of peak
height from a standard curve. Percentages of individual metabolites are
expressed on the basis of the total metabolites produced by the isoform or preparation in question.
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-estradiol, the mobile phase was 60%
H2O and 40% acetonitrile. TST and
17-estradiol were eluted isocratically at a flow rate of 1.0 ml/min
for 15 min, separated by a Prodigy column [Prodigy 3 µ, 150 × 4.6 mm, ODS (3), 100A, Phenomenex, Rancho Palos Verdes, CA] and
detected at 200 nm. The retention time of 17-estradiol and TST was
10.4 and 11.3 min, respectively. The limit of detection for
17-estradiol was approximately 0.10 µM. Concentrations of
metabolites were obtained by extrapolation of peak height from a
standard curve.
Data Analysis and Statistics.
The apparent Km and
Vmax parameters were calculated using
nonlinear regression analysis program (Prism, GraphPad software Inc.,
San Diego, CA), and the Ki values were
estimated by nonlinear regression analysis from the Dixon plot (Segel,
1975) using SigmaPlot Enzyme Kinetics Module (Chicago, IL). Significant
differences between data sets were determined by one-way analysis of
variance, and multiple comparisons were performed with the Dunnett's
method using a JMP 4.0.2, SAS program (SAS, 1989).
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Four major metabolites were formed after incubation of TST with
pooled HLM: 6-, 2-, 15OHTST, and 4-OHAD as well as seven minor
metabolites (Fig. 1). Among 16 different
human P450 isoforms screened, only 2C9*3 (Leu359)
had no detectable activity toward TST (Table
2). All other P450 isoforms were active
in generating one or more than one TST metabolites, although the extent
of metabolism and the ratios of metabolites varied widely among
isoforms. In this comparison of metabolite production by equal
quantities of each isoform, CYP3A4, 3A5, and 3A7 were most active in
TST metabolism among all the P450 isoforms tested (93.5% of the
metabolites produced by all isoforms). Among members of the CYP3A
subfamily, CYP3A4 produced the highest amount of total TST metabolites
(88.5%) compared with 3A5 (6.9%) and 3A7 (4.6%). 6-OHTST, the
most prominent TST metabolite, mainly produced by the CYP3A subfamily,
accounts for 86% of all TST metabolites. Among the CYP3A subfamily,
CYP3A4 produced the highest amount of 6-OHTST (90.6%) compared with 3A5 (7.1%) and 3A7 (2.2%). Other major TST metabolites formed by
CYP3A4 were 15-, 2-OHTST, and 4-OHAD, whereas 6-, 16-, 11-, 2-OHTST, 11-KTST, and AD were minor metabolites. Among the
P450 isoforms tested, CYP3A5 and 3A7 were significantly more important
in forming the major TST metabolites than most of the others, but their
activity was 10- to 20-fold less than that of CYP3A4. Interestingly,
CYP3A7 produced 16 times more 2-OHTST than CYP3A4. CYP1A1 is
involved in the oxidation of TST at the 6-position (3.0 nmol/nmol
isoform/min), whereas CYP1A2 oxidized TST poorly at the 6-position
(0.6 nmol/nmol isoform/min). As can be observed in Table 2, the other
P450 isoforms tested generally produced small amounts of one or more
TST metabolites. CYP2C19 metabolized TST to AD more actively than any
other isoform tested, whereas it catalyzed the formation of 6-,
6-, 16-, 16-, and 2-OHTST poorly.
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HLM and CYP3A4 displayed similar Km
values for 6-, 2-, and 15-OHTST (Table
3). Vmax
and intrinsic clearance rate [Clint (Vmax/Km)]
for 6-OHTST was significantly higher than 2-OHTST (~18-fold)
and 15-OHTST (~40-fold), respectively.
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The effects of various deployment-related chemicals on TST metabolism
were investigated by preincubating them with pooled HLM (Fig. 1).
Preincubation of pooled HLM with chlorpyrifos, phorate, and fonofos
resulted in significant inhibition of 6-, 2-, 15-OHTST, 11-KTST, 11-OHAD, and 4-OHAD. Preincubation of pooled HLM with DEET,
chlorpyrifos-oxon, phorate, imidacloprid, and deltamethrin in some
cases caused small but significant increases in the production of some
TST metabolites by HLM.
Preincubation of CYP3A4 with a variety of chemicals resulted in varying
levels of activation and inhibition of TST metabolism (Fig.
2). The greatest inhibition of TST
metabolism was observed for the organophosphorus compound chlorpyrifos
with up to 98% inhibition of major (6-, 2-, 15-OHTST, and
4-OHAD) and several minor (11-KTST, 16-OHTST, 11-OHAD, and AD)
TST metabolites. However, chlorpyrifos-oxon, an active metabolite of
chlorpyrifos, has no inhibitory effect on the major TST metabolites.
Two other organophosphorus compounds, phorate and fonofos, also
significantly inhibited formation of several TST metabolites including
6-, 2-, 15-OHTST, 11-KTST, 11-OHAD, AD, and 4-OHAD. In
contrast, preincubation of CYP3A4 with pyridostigmine bromide resulted
in the production of small but significantly greater levels of the 6- and 2-OHTST metabolites. Some other TST metabolites were also
significantly increased by preincubation of CYP3A4 with
chlorpyrifos-oxon, phorate, and fonofos.
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To investigate the type of inhibition of CYP3A4 by chlorpyrifos, fonofos, and phorate on major TST metabolites, chlorpyrifos (2 µM), fonofos (5 µM), and phorate (30 µM) were preincubated for 5 min before adding the varying concentrations of TST. Michaelis-Menten plots showed that the Vmax values were significantly reduced without affecting Km values, indicative of a noncompetitive inhibition of major TST metabolites by chlorpyrifos (Fig. 3). Similar results were obtained with fonofos and phorate (data were not shown). Further investigation of noncompetitive reversible or nonreversible inhibition data revealed that the inhibition is nonreversible (Fig. 4).
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The Ki, an indicator of inhibitor
affinity to target enzyme, was calculated by Dixon plot (Table
4; Fig. 5).
Chlorpyrifos was the most potent inhibitor of major TST metabolites
with Ki values ranges from 2.0, 3.6, and 3.7 µM for 6-, 2-, 15-OHTST, respectively. Fonofos was
the second best inhibitor with Ki
values ranging from 5.8, 10.1, and 6.3 µM for 6-, 2-,
15-OHTST, respectively. Phorate Ki
values ranged from 34.1, 42.9, and 33.8 µM for 6-, 2-,
15-OHTST, respectively.
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We investigated the possibility that b5
may stimulate CYP3A5 catalytic activity by incubating
b5 (20 pmol) and CYP3A5 (10 pmol), which,
in the preparations used, does not have b5
coexpressed, with 250 µM of TST for 10 min. Addition of
b5 resulted in a more than 2-fold increase
in TST 6- and 2-OHTST activity.
The possibility that conversion of TST to estradiol, which is catalyzed by aromatase (CYP19), could be inhibited by the test compounds was also investigated. Preincubation of human aromatase (CYP19) with various chemicals (chlorpyrifos, chlorpyrifos-oxon, permethrin, pyridostigmine bromide, DEET, phorate, fonofos, fipronil, imidacloprid, and deltamethrin) had no significant effect on the production of estradiol (data not shown). However, incubation with 4-OHAD, a well known competitive aromatase inhibitor, resulted in 90% inhibition of the aromatase enzyme activity.
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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P450-dependent hydroxylation appears to be a major pathway of
oxidative metabolism of TST in mammalian liver. Studies carried out
using human P450 isoforms provide further insight into the range of TST
hydroxylation reactions that can be catalyzed by human P450 enzymes.
Our isoform data corroborates earlier findings (Waxman et al., 1988,
1991; Yamazaki and Shimada, 1997) that CYP3A4 is one of the major
isoforms responsible for TST metabolism, and 6-OHTST is the major
TST metabolite. Greater than 82% of the TST metabolites are formed by
CYP3A4, and 87% of the major 6-OHTST metabolite is formed by
CYP3A4. The mean metabolic intrinsic clearance rates, as estimated by
Vmax/Km,
also indicated that 6-OHTST is the major metabolite of TST.
Interestingly, CYP3A4 also metabolized TST to 4-OHAD, a potent
inhibitor of extrahepatic aromatase (CYP19). It has been reported that
4-OHAD was able to inhibit 90% of the aromatase activity at a
concentration of 1 µM (Mak et al., 1999). The physiological
significance or consequence of this reaction is unclear and will
require further investigation. Our results indicate that CYP1A1 and 1A2
were able to metabolize TST to 6-OHTST, however, activity of CYP1A1
was much higher (4.7-fold) than CYP1A2. Consistent with a previous
report (Yamazaki and Shimada, 1997), our data also indicated that
CYP2C19 catalyzed oxidation of TST to form AD as a major TST
metabolite. However, CYP2C18, which has 81% amino acid sequence
identity to CYP2C19, exhibited distinctly poor hydroxylation activity
in comparison with CYP2C19. Furthermore, our data indicated that
CYP2D6*1, 4A11, and 2A6 metabolized TST to form AD but not as actively
as CYP2C19. Guengerich et al. (2002) characterized the affinity of
CYP2D6 for testosterone.
Endogenous steroids, such as TST, always exist in vivo, and
considerable amounts of these steroids are metabolized by the P450s
expressed in the human liver, where foreign compounds are mainly
metabolized. If xenobiotics substantially affect TST metabolism, it may
alter the rate of TST metabolism, which may ultimately disrupt TST
homeostasis. Preincubation of pooled HLM with organophosphorus compounds, such as chlorpyrifos, phorate, and fonofos, resulted in the
extensive inhibition of major and some minor TST metabolites. Chlorpyrifos, fonofos, and phorate inhibited major TST metabolites noncompetitively and irreversibly, and it is clear that
organophosphorus compounds are some of the most potent inhibitors of
the CYP3A4-dependent oxidation of TST yet described. Organophosphorus
pesticides, such as chlorpyrifos, phorate, and fonofos are activated by
a P450-catalyzed desulfuration reaction (Fukuto, 1990). The sulfur atom
released from these pesticides in this reaction is highly reactive and is believed to bind immediately to the heme iron of P450 and inhibit its activity (Neal, 1980). On the other hand, enzyme stimulation is a
process by which direct addition of one chemical to an enzyme stimulates the rate of reaction of the substrate (Guengerich, 1997).
Our data indicated that some compounds, such as pyridostigmine bromide,
DEET, chlorpyrifos-oxon, phorate, imidacloprid, and deltamethrin may
stimulate the production of some of the TST metabolites.
Several studies, including this, have shown that CYP3A4 is the major
P450 involved in the metabolism of TST in human liver microsomes
(Waxman et al., 1988, 1991; Yamazaki and Shimada, 1997). Either
inhibition or induction can modulate the activity of an enzyme; P450s
may exhibit stimulation or inhibition in the presence of certain
xenobiotic compounds (Guengerich, 1997; Szklarz and Halpert, 1998). It
has been suggested that CYP3A4 is an allosteric enzyme, even though the
identity of the allosteric site is not known (Shimada and Guengerich
1989; Lee et al., 1995). In addition, little is known about the active
site topology of CYP3A4, although it is generally recognized that the
active site of this enzyme has the capacity to accommodate large
molecules and even more than one substrate (Shou et al., 1994).
Inhibition may, in some interactions, be more serious than enzyme
induction since inhibition happens more rapidly, not taking time to
develop, as with induction (Guengerich, 1997). Preincubation of CYP3A4
with chlorpyrifos resulted in almost complete inhibition of major TST
metabolites. The Ki value indicated
that chlorpyrifos is one of the most potent inhibitors yet shown for
the production of major TST metabolites. This inhibition was not due to
inhibition by the metabolite, chlorpyrifos-oxon, since the latter had
no inhibitory effect on the production of the major TST metabolites.
Phorate and fonofos also inhibited the production of major and some
minor metabolites of TST. The Ki value
indicated that fonofos was a much better inhibitor of major TST
metabolites than phorate. The possibility exists that inhibition of
CYP3A4 may lead to higher levels of TST and may alter hormonal
properties. However, in vivo studies are necessary to understand the
impact of these changes. Preincubation with pyridostigmine bromide
resulted in higher production of 6- and 2-OHTST, suggesting
stimulation of CYP3A4. Preincubation with chlorpyrifos-oxon, phorate,
and fonofos with CYP3A4 also resulted in activation of the production
of some TST metabolites. A number of in vivo studies in rodents have
shown that organochlorine pesticides increased the overall rate of TST
metabolism (Cassidy et al., 1994; Wilson and LeBlanc, 1998; Dai et al.,
2001).
Several studies have demonstrated that simultaneous expression of
CYP3A4 and P450 reductase in bacterial or baculovirus-based insect cell
membranes can produce high catalytic activity for TST 6-OHTST in the
absence of b5 (Guengerich and Johnson
1997; Shaw et al., 1997), although addition of
b5 to the system can enhance the reaction
rates (Yamazaki et al., 1999). In contrast to the CYP3A4 used in these
experiments, cytochrome b5 was not coexpressed in CYP3A5. A comparison of CYP3A5 with and without the
addition of exogenous b5 demonstrated a
2-fold increase in the activity of 6- and 2-OHTST in the presence
of b5.
Human aromatase (CYP19), an extrahepatic P450, catalyzes the conversion
of TST via three hydroxylation steps to estradiol. Inhibitors of
aromatase currently in use have received considerable attention as
treatments for postmenopausal breast cancer and other estrogen-dependent diseases (Bordie et al., 1999). Endocrine disruptors are hormone mimics that modify hormonal action in humans. Currently, inhibitors of human aromatase have been identified as potential endocrine disruptors or environmental toxicants (Mak et al., 1999). The
chemicals used in this study have no significant effect on the activity
of aromatase.
In conclusion, the hydroxylation of TST by P450 isoforms indicates important functions for these enzymes other than detoxification of xenobiotics. The present study provided further insight into the range of TST hydroxylation reactions that can be catalyzed by different human P450 isoforms. The deployment-related chemicals used in this study, including pesticides, caused a marked modification of P450-mediated TST metabolism in vitro. Organophosphorus pesticides were very potent inhibitors of the production of the primary metabolites of CYP3A4 and inhibited major TST metabolites noncompetitively and irreversibly. Addition of b5 to CYP3A5 increased the catalytic activity of this enzyme. Preincubation of the test chemicals had no effect on the production of estradiol from TST.
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Footnotes |
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Received October 16, 2002; accepted December 18, 2002.
This research was supported by US Army Cooperative Agreement DAMD 17-00-2-0008. Part of this study will be presented at the 11th North American ISSX meeting in Orlando, 2002.
Address correspondence to: Ernest Hodgson, Department of Environmental and Molecular Toxicology, Box 7633, North Carolina State University, Raleigh, NC 27695. E-mail: ernest_hodgson{at}ncsu.edu
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Abbreviations |
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Abbreviations used are: P450, cytochrome P450; TST, testosterone; AD, androstenedione; DEET, N,N-diethyl-m-toluamide; KTST, ketotestosterone; OHAD, hydroxyandrostenedione; HPLC, high-performance liquid chromatography; HLM, human liver microsomes; OHTST, hydroxytestosterone; Ki, inhibition constant; b5, cytochrome b5.
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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-hydroxylase cytochrome P-450 enzyme.
Arch Biochem Biophys
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, statistically significantly different
when compared with respective control (P < 0.01).
), 100 µM (
), and 200 µM (
).