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Vol. 28, Issue 3, 348-353, March 2000
Center for Human Toxicology, Department of Pharmacology and Toxicology, University of Utah, Salt Lake City, Utah
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Abstract |
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 Top
 Abstract
 Introduction
Experimental Procedures
Results and Discussion
References
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The cytochrome P450 (P450) 2D subfamily catalyzes ring
hydroxylation of amphetamines. We tested the hypothesis that P450 2D is
selectively involved in amphetamine 4-hydroxylation. Urinary elimination of 4-hydroxyamphetamine and amphetamine was determined in
male Sprague-Dawley rats pretreated with P450 inducers and inhibitors. The urinary 24-h metabolic ratio
(amphetamine/4-hydroxyamphetamine) was not affected by the inducers
3-methylcholanthrene, isosafrole, phenobarbital, ethanol,
pregnenolone-
-carbonitrile, and clofibrate. Isosafrole did,
however, increase amphetamine elimination along with urine volume.
Urinary elimination of 4-hydroxyamphetamine was significantly decreased
by, and the metabolic ratio increased by, the inhibitors
1-aminobenzotriazole, CCl4, quinidine, quinine, and
primaquine. Diallyl sulfide and troleandomycin had no effect. In rat
liver microsomes primaquine was shown to be an inhibitor of 2D
activity. Urine 4-hydroxyamphetamine content correlated strongly
(r2 = 0.989) with microsomal P450 2D
activity in parallel-treated rats. These studies also substantiate that
4-hydroxylation of amphetamine is selectively performed by the P450 2D
subfamily in the rat.
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Introduction |
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Top
Abstract
Introduction
Experimental Procedures
Results and Discussion
References
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Hydroxylation of the aromatic
ring, or its substituents, is a major route of metabolism of
amphetamines. Hydroxylation at either the para- or
meta- position is favored by cytochrome P450 (P450)2
2D3
because these positions are 5 to 7 Angstroms from the basic nitrogen of
the amphetamines (de Groot et al., 1997
). The involvement of P450 2D in
ring hydroxylation of amphetamines in rats and humans has been
substantiated experimentally with poor metabolizers, specific
inhibitors of 2D, and in vitro metabolism systems. The list of
amphetamines that are ring-hydroxylated by P450 2D includes: 1)
amphetamine (Moody et al., 1990; Law and Moody, 1994; Wu et al., 1997);
2) methamphetamine (Lin et al., 1995, 1997); 3) 4-methoxyamphetamine (Kitchen et al., 1979; Wu et al., 1997); 4) 2-methoxyphenamine (Roy et
al., 1985; Coutts et al., 1994); 5) methylenedioxymethamphetamine (MDMA) (Kumagai et al., 1994; Lin et al., 1997); and 6) 6-OH-MDMA (Lim
and Foltz, 1991). Although these studies have thoroughly demonstrated
that the P450 2Ds in both the rat and human catalyze ring hydroxylation
of amphetamines, the selectivity of the involvement of P450 2D has
rarely been addressed. A single study (Kumagai et al., 1994) has
examined the coordinated response of P450 2D and ring hydroxylation of
an amphetamine, MDMA, to three P450 inducers. At this time, we have
extended our studies on the in vivo 4-hydroxylation of amphetamine in
the rat. We used isozyme-selective inducers and inhibitors of P450s and
broad spectrum P450 inhibitors to test for selectivity in the
4-hydroxylation of a single dose of amphetamine. These findings were
also compared with the hepatic response of P450 2D activity.
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Experimental Procedures |
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Top
Abstract
Introduction
Experimental Procedures
Results and Discussion
References
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Materials.
Reagents were purchased from the following: isosafrole, 4-nitrophenol,
and erythromycin sulfate, Aldrich Chemical Co. (Milwaukee, WI);
3-methylcholanthrene and triethylamine, Eastman Chemical Co.
(Rochester, NY); 14C-lauric acid, ICN (Irvine,
CA); 3H-dextromethorphan, E. I. DuPont de
Nemours and Company (Billerica, MA); ethoxyresorufin and
pentoxyresorufin, Pierce (Rockford, IL); opti-fluor scintillation
cocktail, Packard Instrument Co., Inc. (Meriden, CT); ethanol (100%),
Quantum Chemical Corp. (Tuscola, IL); sodium phenobarbital,
pregnenalone--carbonitrile (PCN), clofibrate, 1-aminobenzotriazole,
quinidine sulfate, quinine hydrochloride, primaquine diphosphate,
diallyl sulfide, d-amphetamine sulfate, d-methamphetamine hydrochloride, troleandomycin, lauric
acid, NADPH, glucose 6-phosphate, glucose 6-phosphate dehydrogenase, 
-glucuronidase (Type H-1), and dextromethorphan hydrobromide, Sigma
Chemical Co. (St. Louis, MO); Bond Elut C18 columns, Varian (Harbor
City, CA); and CCl4 and HPLC grade solvents,
Burdick and Jackson (Muskegon, MI). All other reagents were of reagent
grade or better. Dextrorphan tartrate was a gift from Dr. Peter Sorter, Hoffman-La Roche, Inc. (Nutley, NJ). 4-Hydroxyamphetamine hydrobromide and 4-hydroxymethamphetamine were gifts from Dr. Anthony S. Murabito of
SmithKline Beecham (Philadelphia, PA).
Animals. Male Sprague-Dawley (SD) rats weighing 180 to 220 g (Sasco, Inc., Omaha, NE) were housed in an environmentally (humidity- and temperature-) controlled room with a 12-h light/dark cycle. The rats were acclimated in metal drawer type cages (four per cage) for at least 5 days on arrival, with food and water available ad libitum. At the end of the acclimatizing period, the animals were treated with P450 modulators and those challenged with d-amphetamine sulfate were placed individually in Nalgene (Rochester, NY) metabolic cages for urine collection (see below). Rats used for microsome preparation remained in the metal drawer cages, and all rats continued to receive food and water ad libitum. All of the experimental protocols were approved by the University of Utah Institutional Animal Care and Use Committee.
Treatments. Rats were given inducers as follows: 3-methylcholanthrene (daily 25 mg/kg in corn oil, i.p., 3 days); isosafrole (daily 150 mg/kg in corn oil, i.p., 4 days); clofibrate (daily 300 mg/kg in corn oil, i.p., 5 days); PCN (daily 50 mg/kg in corn oil, i.p., 3 days); phenobarbital (0.1% of drinking water, 5 days); and ethanol (15% of drinking water, 5 days). Rats were given inhibitors as follows (times listed are the time before the amphetamine dose): 1-aminobenzotriazole (100 mg/kg in saline, i.p., 2 h); quinidine (80 mg/kg in saline, i.p., 4 h); quinine (25 mg/kg in saline, i.p., 4 h); primaquine (25 mg/kg in saline, i.p., 4 h); diallyl sulfide (200 mg/kg in corn oil, i.p., 2 h); troleandomycin (500 mg/kg in corn oil, i.p., 24 h); and CCl4 (0.05 ml/kg in mineral oil, i.p., 4 h). CCl4 and the mineral oil controls were given at 0.5 ml/kg to phenobarbital-pretreated rats. All other i.p. injections were at 1.0 ml/kg and rats had no other pretreatment. Rats were grouped for treatment based on the vehicle used for delivery of the modulator. With each group was a set of controls (n = 3 or 4 for urine collection, and n = 2 or 3 for microsome preparation) that received the same vehicle. When different treatments were administered for different times (e.g., clofibrate for 5 days, isosafrole for 4 days), the control rats were injected with vehicle for the longer duration. One day after either the last dose of inducer, or at the specified time after an inhibitor, a set of animals (n = 3 or 4) was challenged with an i.p. dose of 10 mg/kg of d-amphetamine sulfate in normal saline, and placed in metabolic cages, one per cage, for urine collection at 12-h intervals for 2 days. The other set of rats (n = 2 or 3) were euthanized by carbon dioxide asphyxiation for liver microsome preparation as described below. All rats were weighed daily, and urinary pH values (by pH indicator strip; Baxter Healthcare Corp., McGaw Park, IL) and volumes were recorded throughout the experimental period.
Urine Analysis.
The rat urine samples were analyzed using a previously described method
(Law and Moody, 1995). Briefly, the urine samples were first hydrolyzed
with -glucuronidase, and the 4-hydroxyamphetamine and amphetamine
were extracted using C18 Bond Elut columns. The amount of
4-hydroxyamphetamine and amphetamine in urine samples were determined
using a UV detector set at 215 nm in an HPLC system (Varian, Walnut
Creek, CA) with a Microsorb phenyl column (Rainin, Emeryville, CA).
Microsome Preparation.
Microsomes were prepared by differential centrifugation of homogenates
of perfused livers as described previously (Moody et al., 1981). The
final pellets were resuspended in 0.1 M phosphate buffer with 10%
glycerol (pH 7.4) and samples were taken for protein determination as
described by Lowry et al. (1951) with BSA as standard. The microsome
samples were adjusted to 30 mg of protein/ml and aliquots were stored
at 
70°C until use.
Microsomal Monooxygenase Assays.
Microsomal monooxygenase activities dependent on P450 1A and 2B were
determined spectrophotometrically as ethoxyresorufin O-deethylation (EROD) and pentoxyresorufin
O-dealkylation (PROD), respectively (Klotz et al., 1984;
Lubet et al., 1985). P450 2D-dependent activity was determined from
dextromethorphan O-demethylation to dextrorphan using our
previously described radiometric-thin layer chromatographic method
(Singh and Moody, 1995). P450 2E-dependent activity was estimated by
the formation of 4-nitrocatechol in the 4-nitrophenol hydroxylation
reaction (Papac and Franklin, 1988). The activity of P450 3A was
estimated by the amount of formaldehyde produced during erythromycin
N-demethylation (Nash, 1953) using the conditions described
by Papac and Franklin (1988). P450 4A-dependent activity was determined
by selective solvent partition of 14C-lauric acid
from radiolabeled 11- or 12-hydroxylauric acid as described by Giera
and van Lier (1991).
Microsomal P450 2D Activity Inhibition Study. The in vitro effects of quinine, quinidine, and primaquine on P450 2D were determined using the radiometric dextromethorphan O-demethylation assay. The assay was carried out in liver microsomes from untreated rats (n = 2) as described above except that 50 µl of inhibitors in water were added to achieve final concentrations of 0, 5, 25, 50, 250, and 500 µM 5 min before initiating the O-demethylation reaction by the addition of the NADPH-generating system.
Data Handling and Statistics.
Descriptive statistics (mean, S.D., S.E.M., one-way ANOVA) were
calculated by Microsoft EXCEL (Redmond, WA). Treated rats were compared
with grouped controls. One-way ANOVA was used (P < .05) with the Tukey posthoc test (P < .05) (Zar,
1984). Correlation coefficeints (r2)
were determined using plots of relative changes as described previously
(Moody et al., 1981), except that means of urine values were compared
with means of microsomal values as different animals were used for the
respective experiments. Regression equations were determined by CRICKET
GRAPH (CA Associates, Malvern, PA).
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Results and Discussion |
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Top
Abstract
Introduction
Experimental Procedures
Results and Discussion
References
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Controls and Expression of Results.
The primary endpoint for determination of in vivo hydroxylation of
amphetamine was the 24-h metabolic ratio (molar amphetamine divided by
molar 4-hydroxyamphetamine). This is a well established endpoint to
assess P450 2D activity in vivo when sufficient parent compound is
excreted (Horai et al., 1990), as is the case with amphetamine. As the
compounds used to modulate P450s potentially could affect other
non-P450 factors regulating urinary elimination, the response of the
individual components of the metabolic ratio, urinary amphetamine and
4-hydroxyamphetamine, have been presented. The combined amounts of
amphetamine and 4-hydroxyamphetamine were also determined to rule out
changes in alternative pathways (e.g., N-oxidation) that
could have an effect on the metabolic ratio. In both the induction and
inhibition studies, variations between control groups were seen in the
excretion of amphetamine (4.2-fold), 4-hydroxyamphetamine (3.2-fold),
the combined total of amphetamine and 4-hydroxyamphetamine (2.6- fold),
and the metabolic ratio (4.8-fold). These variations could not be
explained by vehicle used. Therefore, in assessment of the following
induction and inhibition studies, treated animals were compared with
controls that were matched by vehicle and time of treatment.
P450 Induction Studies.
To validate the induction dosing regimens, isozyme-selective
monooxygenase activities were measured in liver microsomes from rats
receiving the same treatments. EROD activity (P450 1A) was only
detectable in rats treated with 3-methylcholanthrene and isosafrole.
PROD activity (P450 2B) was only detectable in rats treated with
phenobarbital. Dextromethorphan O-demethylation activity (P450 2D) was not significantly changed. Ethanol and phenobarbital increased 4-nitrophenol hydroxylation (P450 2E) 2.6- and 1.3-fold, respectively. Erythromycin N-demethylation (P450 3A), was
increased by PCN 1.7-fold and phenobarbital 1.6-fold. The peroxisome
proliferator, clofibrate, increased lauric acid hydroxylation (P450 4A)
activity 7.9-fold. Phenobarbital, PCN, and ethanol increased the
activity 3-, 2.3-, and 2-fold, respectively, consistent with the
ability of this assay to also detect 
-1 hydroxylation (Giera and van Lier, 1991). These results substantiate the dosage, route of
administration, and the duration of induction as appropriate.
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; Yamamoto et al., 1984).
Our studies extend findings on the noninducibility of amphetamine
4-hydroxylation to inducers of P450s 1A2, 2E, and 4A. Our in vitro
measurements of dextromethorphan O-demethylation responses
to inducers are consistent with those of Matsunaga and coworkers (1989)
that rat liver P450 2D was not induced by phenobarbital, 3-methylcholanthrene, or dexamethasone. This noninducibility of P450 2D
activity in rat liver has now been extended to isosafrole, ethanol,
PCN, and clofibrate.
P450 Inhibition Studies.
4-Hydroxyamphetamine excretion was significantly lowered in rats
pretreated with 1-aminobenzotriazole, CCl4,
quinidine, quinine, and primaquine (Fig.
2A). A converse increase in amphetamine
excretion was observed in rats pretreated with 1-aminobenzotriazole,
CCl4, quinidine, and primaquine (Fig. 2B). Only
CCl4 caused a significant change in the sum of
amphetamine and 4-hydroxyamphetamine excreted (a 2-fold increase) (Fig.
2C). This could be due to the nephrotoxicity of
CCl4 (Striker et al., 1968). No changes in
amphetamine or 4-hydroxyamphetamine excretion were observed in rats
pretreated with the P450 2E inhibitor, diallyl sulfide (Chen and Yang,
1996), or the P450 3A inhibitor, troleandomycin (Fisher et al., 1990).
A dramatic increase was found in the metabolic ratios of rats
pretreated with the effective inhibitors (Fig. 2D).
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) and
CCl4 (Moody et al., 1986) have both been found to
irreversibly inhibit a number of P450s within rat liver microsomes.
Until now, however, their effect on P450 2D has not been examined. In
rat liver microsomes prepared at the time amphetamine was administered
to other rats, we found dextromethorphan O-demethylation
activity reduced to 44 and 69% of their controls, respectively. This
clearly shows that these two inhibitors do affect P450 2D.
In the rat, quinine is known to be 10 to 50 times more potent as an in
vitro inhibitor of liver microsomal P450 2D than its diastereoisomer,
quinidine (Kobayashi et al., 1989) (see also below). We show that the
relative potency does not hold true under in vivo conditions.
Quinidine, at 80 mg/kg, was a more potent in vivo inhibitor of
amphetamine 4-hydroxylation than quinine at 25 mg/kg. This is
consistent with the findings of Muralidharan et al. (1991) who used the
same doses to explore inhibition of P450 2D mediated
O-demethylation and 5-hydroxylation of methoxyphenamine. The
reason for the in vitro and in vivo differences in quinine and
quinidine inhibition of rat liver P450 2D have not yet been explained.
They do not appear to arise from differences in rates of
biotransformation, because similar half-lives were observed after i.v.
(Watari et al., 1989) administration of quinine and quinidine to rats.
Primaquine was used in this study because it is a reported inhibitor of
in vivo tolbutamide hydroxylation in rats (Back et al., 1984).
Primaquine, however, significantly lowered the urinary excretion of
4-hydroxyamphetamine. As we did not measure the response of
dextromethorphan O-demethylation in primaquine-treated rats, we performed a subsequent study to measure the in vitro effect of
primaquine on rat liver microsomal dextromethorphan
O-demethylation. Quinine and quinidine were studied as
positive controls. Primaquine caused a concentration-dependent decrease
in dextromethorphan O-demethylation with an apparent
IC50 of 40.9 µM. The corresponding IC50 values for quinine and quinidine were 4.7 and 30.3 µM, respectively. Therefore, all the inhibitors of in vivo
4-hydroxylation of amphetamine have been found to effectively inhibit
P450 2D activities in the rat, and the inhibition studies confer the
selectivity of 2D in this pathway.
In Vivo and In Vitro Correlations. Additional rats were concurrently treated with the inducers (as mentioned above) and most of the inhibitors to compare their in vivo effect with the response of P450 isozymes in the liver. Liver microsomes were prepared and assayed for the monooxygenase activities described above, except for EROD and PROD which were only detectable after certain inductions. The relative change (ratio of treated mean to control mean) in microsomal monooxygenase activities was plotted in scattergrams versus the relative change in urine amphetamine and 4-hydroxyamphetamine. Linear regression lines and correlation coefficients (r2) were determined for rats treated with only the inhibitors, only the inducers or all treatments combined (see Fig. 3 for a representative scattergram). Correlations of in vivo urine amphetamine and 4-hydroxyamphetamine with the four in vitro monooxygenase activities are shown in Table 1.
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Conclusions.
The purpose of the current study was to demonstrate the effects of
modulating P450s in rats on amphetamine 4-hydroxylation to establish
isozyme selectivity in this metabolic pathway. The induction and
inhibition studies showed that the P450 isozyme(s) involved in
amphetamine 4-hydroxylation is not inducible, but is diminished by P450
2D inhibitors. The positive correlation of urinary 4-hydroxyamphetamine
excretion with liver microsomal P450 2D activity further supports the
idea that amphetamine 4-hydroxylation is primarily mediated by P450 2D
in the rat. Although humans use other pathways in addition to
4-hydroxylation in the elimination of amphetamine, a known P450 2D6
poor metabolizer, however, did report an adverse response to ingestion
of a single dose of amphetamine (Smith, 1986). As more sympathomimetic
phenylalkylamines are found in weight control products, more humans are
exposed to the dangers of overdose with these compounds. Understanding
the metabolism of amphetamines is useful to identify those who are
genetically predisposed to dysfunctional metabolism. In these studies,
we were able to isolate and study one of the amphetamine metabolic pathways, 4-hydroxylation. Future studies should include other pathways
to understand their regulation of amphetamine metabolism.
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Footnotes |
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Received April 23, 1999; accepted December 8, 1999.
1 Current address: Chattem, Inc., 1715 West 38th Street, Chattanooga, TN 37409.
This work was supported in part by United States Public Health Service Grants DA05102 and DA10100.
3
The metabolic activity of P450 2D in human liver
is the result of expression of a single gene product, P450 2D6;
however, activity in the rat results from expression of four genes,
2D1, 2D2, 2D3, and
2D5 (Matsunaga et al., 1989). As most of the work reported or cited in this paper does not distinguish a specific rat
gene product, the subfamily term P450 2D will be used when discussing
the rat enzymes.
Send reprint requests to: David E. Moody, Ph.D., University of Utah, Center for Human Toxicology, 20 S 2030 E, Rm. 490, Salt Lake City, Utah 84112-9457. E-mail: dmoody{at}alanine.pharm.utah.edu
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Abbreviations |
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Abbreviations used are:
P450, cytochrome P450;
SD, Sprague-Dawley;
MDMA, methylenedioxymethamphetamine;
PCN, pregnenolone--carbonitrile;
EROD, ethoxyresorufin
O-deethylation;
PROD, pentoxyresorufin
O-dealkylation.
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References |
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Top
Abstract
Introduction
Experimental Procedures
Results and Discussion
References
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-oxidation based on solvent partition.
Fundam Appl Toxicol
16:
348-355[Medline].
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) 3-MC,
3-methylcholanthrene; PhBarb, phenobarbital; EtOH, ethanol. *,
significantly different from matched controls, P < .05.
, - - -, r2 = 0.989), inducers (
,
····, r2 = 0.457), or all
treatments (
, 0.758).