Cytochrome P4502D and -2C enzymes catalyze the oxidative N-demethylation of the Parkinsonism-inducing substance 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in rat liver microsomes

Article abstract of DOI:10.1021/tx9500540

We have examined the oxidative N-demethylation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a Parkinsonism-inducing neurotoxin, in liver microsomes from adult Wistar and Dark Agouti (DA) rats. The oxidation of MPTP to 4-phenyl-1,2,3,6-tetrahydropyridine (PTP) in these preparations required NADPH as a cofactor and was significantly inhibited by SKF 525-A (2 mM). MPTP N-demethylation exhibited biphasic kinetics, consistent with two enzymes, a low K_m system (K_m1, 10.0 ± 2.2 μM; V_max1, 0.048 ± 0.009 nmol/(min·mg of protein)) and a high K_m system (Km₂, 1180 ± 91 μM; V_max2, 4.80 ± 0.75 nmol/(min·mg of protein)). We thus employed two substrate concentrations, 5 μM and 5 mM, for the low and high K_m system, respectively, to assay enzyme activity in subsequent experiments. The oxidation activity was significantly decreased by pretreatment of rats with phenobarbital and β-naphthoflavone. Furthermore, marked strain (Wistar > DA) and sex (male > female) differences were observed at low (5 μM) and high (5 mM) substrate concentrations, respectively. Reconstitution experiments with cytochrome P450BTL, which belongs to the P4502D subfamily, and P450ml (P4502C11) demonstrated that MPTP N-demethylase occurs at concentrations of 5 μM and 5 mM. At 5 mM the male-specific P450ml showed a remarkably high activity, over 400-fold that of P450BTL. Polyclonal antibodies against P450BTL and P450ml effectively suppressed the activity at the low (5 μM) and the high (5 mM) substrate concentrations, respectively. These results suggest that, in the microsomal preparations used, MPTP N-demethylation is mainly mediated by P4502D enzyme(s) at lower substrate concentrations and by P4502C11 at higher substrate concentrations.

Full text of DOI:10.1021/tx9500540

Chem. Res. Toxicol. 1996, 9, 93-98
93
Cytoch r om e P 4502D a n d -2C En zym es Ca ta lyze th e
Oxid a tive N-Dem eth yla tion of th e
P a r k in son ism -In d u cin g Su bsta n ce
1
-Meth yl-4-p h en yl-1,2,3,6-tetr a h yd r op yr id in e in Ra t Liver
Micr osom es
Shizuo Narimatsu,* Masaya Tachibana, Yasuhiro Masubuchi, and Tokuji Suzuki
Laboratory of Biopharmaceutics, Faculty of Pharmaceutical Sciences, Chiba University,
1
-33 Yayoi-cho, Inage-ku, Chiba 263, J apan
Received March 20, 1995X
We have examined the oxidative N-demethylation of 1-methyl-4-phenyl-1,2,3,6-tetrahydro-
pyridine (MPTP), a Parkinsonism-inducing neurotoxin, in liver microsomes from adult Wistar
and Dark Agouti (DA) rats. The oxidation of MPTP to 4-phenyl-1,2,3,6-tetrahydropyridine
(
PTP) in these preparations required NADPH as a cofactor and was significantly inhibited by
SKF 525-A (2 mM). MPTP N-demethylation exhibited biphasic kinetics, consistent with two
enzymes, a low K system (Km1, 10.0 ( 2.2 µM; Vmax1, 0.048 ( 0.009 nmol/(min‚mg of protein))
and a high K system (Km2, 1180 ( 91 µM; Vmax2, 4.80 ( 0.75 nmol/(min‚mg of protein)). We
thus employed two substrate concentrations, 5 µM and 5 mM, for the low and high K system,
m
m
m
respectively, to assay enzyme activity in subsequent experiments. The oxidation activity was
significantly decreased by pretreatment of rats with phenobarbital and â-naphthoflavone.
Furthermore, marked strain (Wistar > DA) and sex (male > female) differences were observed
at low (5 µM) and high (5 mM) substrate concentrations, respectively. Reconstitution
experiments with cytochrome P450BTL, which belongs to the P4502D subfamily, and P450m1
(P4502C11) demonstrated that MPTP N-demethylase occurs at concentrations of 5 µM and 5
mM. At 5 mM the male-specific P450m1 showed a remarkably high activity, over 400-fold
that of P450BTL. Polyclonal antibodies against P450BTL and P450m1 effectively suppressed
the activity at the low (5 µM) and the high (5 mM) substrate concentrations, respectively.
These results suggest that, in the microsomal preparations used, MPTP N-demethylation is
mainly mediated by P4502D enzyme(s) at lower substrate concentrations and by P4502C11 at
higher substrate concentrations.
In tr od u ction
1
-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)1
is a precursor to a Parkinsonism-inducing neurotoxic
agent (1, 2). The compound easily passes through the
blood-brain barrier and is oxidized to 1-methyl-4-phenyl-
+
2
,3-dihydropyridinium ion (MPDP ) by monoamine oxi-
+
dase B in brain tissues (Figure 1). MPDP is then
nonenzymatically converted to the toxic compound
N-methyl-4-phenylpyridinium ion (MPP ), which, after
+
being taken up into dopaminergic neurons, causes neu-
ronal toxicity in experimental animals and humans (3-
5
).
MPTP is also oxidized by the microsomal flavin-
containing monooxygenase (FMO) to MPTP N-oxide, and
this pathway was thought to be the major detoxication
pathway (6-8). Cytochrome P450 (P450) has been
reported to be involved in other oxidative pathways of
MPTP such as N-demethylation to 4-phenyl-1,2,3,6-
tetrahydropyridine (PTP) (6, 8), a nontoxic metabolite (9),
+
and oxidative dehydrogenation forming MPDP and
+
MPP (10) (Figure 1). Accumulated knowledge indicates
F igu r e 1. Oxidative metabolism of MPTP in the liver. MAO-
B, monoamine oxidase B.
*
Author to whom correspondence should be addressed.
X
Abstract published in Advance ACS Abstracts, December 1, 1995.
Abbreviations: MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyri-
+
that MPTP is primarily converted to MPDP by monoam-
1
+
ine oxidase B, and contribution of P450 forming PTP and
FMO forming MPTP N-oxide to the overall MPTP
biotransformation is limited in the peripheral system,
especially in the liver (6, 7, 11). However, the formation
dine; PTP, 4-phenyl-1,2,3,6-tetrahydropyridine; MPDP , 1-methyl-4-
phenyl-2,3-dihydropyridinium ion; MPP , N-methyl-4-phenylpyridin-
ium ion; FMO, flavin-containing monooxygenase; P450, cytochrome
P450; DA, Dark Agouti; SD, Sprague-Dawley; G-6-P, glucose 6-phos-
phate.
+
0
893-228x/96/2709-0093$12.00/0 © 1996 American Chemical Society
+
+

9
4
Chem. Res. Toxicol., Vol. 9, No. 1, 1996
Narimatsu et al.
of PTP is thought to be only one oxidative detoxication
pathway of MPTP because after direct administration to
mouse brain, MPTP N-oxide, which had been believed
to be the major detoxicated metabolite, exerted a neuro-
toxicity similar to MPTP by causing a decrease of
dopamine in nigrostriatal neurons (12).
of G-6-P dehydrogenase, 10 mM MgCl
2
, 1 mg/mL microsomal
protein, 5 µM MPTP, and 154 mM potassium phosphate buffer
pH 7.4). After preincubation at 37 °C for 5 min, incubation
(
was started by adding NADPH (1 mM) and continued for 2.5
min. The reaction was terminated by adding ice-cold 1 N NaOH
to the mixture and cooling at 0 °C in an ice bath. The mixture
was then extracted with 5 mL of ethyl acetate by vigorous
shaking, followed by centrifugation at 3000g for 10 min. To 4
mL of the organic layer were added an internal standard
(flurazepam, 200 ng) and 0.2 mL of 0.01 N HCl. MPTP, PTP,
and the internal standard in the organic layer were then back-
extracted into the aqueous layer. After centrifugation, the
organic layer was discarded by aspiration and the aqueous layer
evaporated with a rotary evaporator. The residue was dissolved
in 100 µL of an HPLC mobile phase described below. The
quantities of PTP were determined by the HPLC from calibra-
tion curves prepared by adding known amounts of synthetic PTP
to ice-cold reaction medium containing the same ingredients
described above, and by extraction without incubation.
Recon stitu tion of MP TP N-Dem eth yla tion Usin g P u r i-
fied P 450 En zym es. P450BTL, a member of the P4502D
subfamily, was purified from liver microsomes of adult male
Sprague-Dawley (SD) rats by following bunitrolol 4-hydroxylase
activity as reported previously (21). This enzyme showed much
higher activities toward bunitrolol and propranolol compared
with 12 other P450 enzymes purified from rat liver in the
reconstituted system, and its N-terminal amino acid sequence
was very similar to that of P4502D2 (21). P450m1 (P4502C11)
was purified from male rats of the same strain as described
elsewhere (22). NADPH-P450 reductase was purified from
liver microsomes of male SD rats pretreated with phenobarbital
according to the method of Yasukochi and Masters (23). The
reconstituted system contained 5 µg of dilauroylphosphatidyl-
choline, 10 mM G-6-P, 1 unit of G-6-P dehydrogenase, 10 mM
There have been several reports on the metabolic
interaction between MPTP and P4502D enzymes (13-
1
6), demonstrating that the compound inhibits P4502D
reactions in humans and rodents. Fonne-Pfister et al.
13, 14) found that bufuralol 1′-hydroxylase, a reaction
(
mediated by P4502D enzymes, was competitively inhib-
ited by MPTP in microsomal fractions from human liver
or rat brain. Among three pathways in which P450 can
be involved (Figure 1), the oxidative dehydrogenation
+
+
pathways forming MPDP and MPP have been shown
to be mediated by rat liver P4502B1 and P4501A1 in the
reconstituted system (9). Thus, the formation of PTP
from MPTP may be catalyzed by P4502D enzymes.
Genetic polymorphism of human P4502D enzyme (2D6)
has been suggested to be associated with susceptibility
to Parkinson’s disease (17-19). In this context, it is of
interest to know whether MPTP is metabolized by
P4502D enzyme(s). However, no systematic studies
clearly demonstrating the participation of P4502D en-
zymes in MPTP N-demethylation have been reported so
far.
The present study was conducted to determine the
P450 enzymes in MPTP N-demethylation in rat liver
microsomes as a detoxication pathway. We have studied
the kinetics of MPTP N-demethylation in this prepara-
tion, and we report here evidence showing that P4502C11
as well as P4502D enzyme(s) is involved in the reaction.
MgCl
, 0.5 unit of NADPH-P450 reductase, MPTP (2.5 nmol
2
or 2.5 µmol), and 154 mM potassium phosphate buffer (pH 7.4)
in a final volume of 0.5 mL. The reaction at 37 °C was started
by adding NADPH (1 mM), allowed to continue for 10 min, and
then terminated by adding 1 N NaOH as described in the
microsomal experiments.
Exp er im en ta l P r oced u r es
Ma ter ia ls. MPTP, PTP, and propranolol as hydrochlorides
were purchased from Sigma Chemical Co. (St. Louis, MO) and
the other compounds obtained as follows: â-naphthoflavone
from Aldrich Chemical Co. (Milwaukee, WI); debrisoquine
hemisulfate from Hoffman La-Roche Co. (Basel, Switzerland);
sodium phenobarbital from Wako Pure Chemical Ind. (Osaka,
J apan); SKF 525-A from Funakoshi Co. (Tokyo, J apan). Other
reagents or chemicals used were of the highest quality com-
mercially available.
An im a ls. Adult Wistar and Dark Agouti (DA) rats of both
sexes (8 weeks old) were obtained from Takasugi Experimental
Animals (Kasukabe, J apan) and SLC (Shizuoka, J apan), re-
spectively, and allowed food and water ad libitum. The animals
were killed by decapitation, and liver microsomes were prepared
according to the method of Omura and Sato (20). In enzyme
induction studies, male Wistar rats were given a daily dose of
phenobarbital or â-naphthoflavone (each 80 mg/kg, ip, for 3
days) and were sacrificed by decapitation 24 h after the final
dose, and liver microsomes were prepared as described above.
In enzyme inhibition studies, the animals were given a daily
dose of propranolol (100 mg/kg, po, for 5 days) and sacrificed
Con d ition s for HP LC a n d LC/MS An a lyses. The HPLC
apparatus consisted of an LC-3A liquid chromatograph equipped
with an SPD-2A UV detector and C-R6A Chromatopac data
processor (Shimadzu, Kyoto, J apan). The chromatographic
conditions were as follows: column, Inertsil ODS (4.6 mm i.d.
× 25 cm, GL Science Co., Tokyo, J apan); column temperature,
40 °C; mobile phase, 50 mM potassium phosphate buffer (pH
7.4)/acetonitrile (55:45 v/v); flow rate, 2.0 mL/min; detection, 244
nm. LC/MS was conducted on a J EOL LX-2000 instrument
under the following conditions: interface, FRIT-FAB; injection,
flow injection (10 µL); matrix, 1% glycerin; collision gas; xenon.
In Vitr o In h ibition Stu d ies. SKF 525-A (a typical inhibitor
of P450, 2 mM) or debrisoquine (a typical substrate of P4502D
enzyme, 50-500 µM) was added to the reaction mixture
containing the same ingredients as mentioned above, except that
the MPTP concentrations used were 1 mM for the SKF 525-A
experiment and 5 µM for the debrisoquine experiment.
Oth er s. Protein concentrations were determined by the
method of Lowry et al. (24). Enzyme kinetic parameters (Km
and Vmax) were analyzed according to a nonlinear least-squares
regression analysis based on a simplex method (25). Statistical
significance was calculated by the Student’s t-test.
4
0 h after the final dose.
Id en tifica tion of P TP by LC/MS. A reaction mixture (5
mL) contained 10 mM glucose 6-phosphate (G-6-P), 10 units of
G-6-P dehydrogenase, 10 mM MgCl
, 10 mg/mL microsomal
protein, 50 µM MPTP, and 154 mM potassium phosphate buffer
pH 7.4). Incubation was started by adding NADPH (1 mM)
2
Resu lts
(
HP LC a n d LC/MS An a lyses of th e F or m a tion of
P TP fr om MP TP in Ra t Liver Micr osom es. In
preliminary experiments, we confirmed that the forma-
tion of PTP increased linearly with time to 5 min and
with microsomal protein concentration to 2 mg/mL.
Based on these results, an incubation time of 2.5 min and
a microsomal protein concentration of 1.0 mg/mL were
used in subsequent experiments. Chromatogram A in
Figure 2 shows a tracing of MPTP (2 nmol), PTP (2 nmol),
and continued for 15 min. The reaction was terminated by
adding ice-cold 1 N NaOH to the mixture followed by extraction
with ethyl acetate (10 mL × 2). The organic layer was dried
2 4
over anhydrous Na SO , and then solvent was removed with a
rotary evaporator. The residue was dissolved in 100 µL of
ethanol as a sample for LC/MS analysis under the conditions
described below.
Assa y of MP TP N-Dem eth yla se Activity by HP LC. A
typical reaction mixture (0.5 mL) contained 10 mM G-6-P, 1 unit
+
+

MPTP N-Demethylation by Rat Liver P450 Enzymes
Chem. Res. Toxicol., Vol. 9, No. 1, 1996 95
F igu r e 3. Typical Eadie-Hofstee plots showing MPTP N-
demethylation in liver microsomes from male Wistar rats. The
substrate concentration range was from 1.25 to 5000 µM.
Kinetic parameters calculated were as follows: Km1 12.9 µM,
F igu r e 2. Typical high-performance liquid chromatograms of
MPTP, PTP, and flurazepam. (A) An ethyl acetate extract of
ice-cold reaction medium containing microsomes, other ingre-
dients, and authentic MPTP (2 nmol), PTP (2 nmol), and
flurazepam (200 ng); (B) an extract of reaction medium contain-
ing microsomes, MPTP (5 nmol), and flurazepam (200 ng); (C)
an ethyl acetate extract of post-incubated reaction medium
containing microsomes, MPTP (5 nmol), and other ingredients
necessary for the monooxygenation.
V
K
K
max1 0.033 nmol/(min‚mg of protein) for a low K
m2 1001 µM, Vmax2 3.44 nmol/(min‚mg of protein) for a high
system.
m
system; and
m
parameters indicated in the legend were obtained. The
mean values of kinetic parameters obtained from three
experiments with different microsomal fractions are as
and the internal standard (flurazepam, 200 ng) which
were added to an ice-cold reaction mixture containing all
the ingredients and extracted without incubation. The
tracing of an extract of a reaction mixture which was
incubated without MPTP but to which MPTP (5 nmol)
and flurazepam had been added just before extraction is
shown as chromatogram B. Chromatogram C is a tracing
of an extract from a reaction mixture in which MPTP (5
nmol) was incubated with an NADPH-generating system.
The retention times of MPTP, PTP, and the internal
standard were 7.0, 3.6, and 12.4 min, respectively.
The identification of the peak with a retention time of
follows: a low K
( 0.009 nmol/(min‚mg of protein); a high K
m
system, Km1 10.0 ( 2.2 µM, Vmax1, 0.048
system, Km2
m
1180 ( 91 µM, Vmax2 4.80 ( 0.75 nmol/(min‚mg of protein)
(mean ( SE of three determinations). These results
indicate that at least two P450 enzymes were involved
in the MPTP N-demethylation in rat liver microsomes.
We therefore employed two substrate concentrations (5
µM and 5 mM for the low and high K
m
systems,
respectively) in subsequent experiments.
Effects of En zym e In d u ction on MP TP N-Dem -
eth yla tion . Figure 4 shows the effects of phenobarbital
or â-naphthoflavone pretreatment of male Wistar rats
on microsomal formation of PTP. Pretreatments with
these inducers reduced N-demethylation activtiy of both
3
.6 min as PTP was further conducted by LC/MS. The
LC/MS analysis of the peak isolated from eluents of the
HPLC showed that a molecular ion (m/z 160, M + H) and
other fragment ions of the peak were in good agreement
with those of a synthetic standard (data not shown). A
peak with a retention time of 2.5 min was thought to be
a mixture of two or three metabolites of MPTP, which
could not be identified from their overlapped and com-
plicated fragment ions. These results showed that MPTP
N-demethylation could be satisfactorily assayed with the
HPLC conditions shown.
m
the low (Figure 4A) and high K (Figure 4B) systems,
with the drop in the activity greater in the â-naphthofla-
vone pretreated animals, particularly at a high substrate
concentration.
Str a in a n d Sex Differ en ces in MP TP N-Dem eth y-
la tion . MPTP N-demethylase activities at a substrate
concentration of 5 µM were significantly lower in DA rats
than in Wistar rats (Figure 4A) and were significantly
higher in male rats of both strains. At a substrate
concentration of 5 mM, the sex difference became more
pronounced (male . female), but there was no strain
difference (Figure 4B).
Con tr ibu tion of P 450 to th e MP TP N-Dem eth y-
la tion in Ra t Liver Micr osom es. To confirm that P450
is responsible for the formation of PTP from MPTP under
the conditions used, we examined the effects of SKF
5
25-A (a typical P450 inhibitor) on the N-demethylase
Effects of En zym e In h ibition on MP TP N-Dem -
eth yla tion . In the course of our studies on the metabolic
activation of propranolol, we have recognized that pre-
treatment of rats with propranolol causes a marked
suppression in various hepatic drug oxidation activities
mediated by P4502D enzyme(s) such as propranolol ring
4-, 5-, and 7-hydroxylase, debrisoquine 4-hydroxylase,
and imipramine 2-hydroxylase activities (26). For this
reason, male Wistar rats were given a daily dose of
propranolol (100 mg/kg, po, for 5 consecutive days). As
shown in Figure 4C, low concentration MPTP N-dem-
ethylase activity in liver microsomes from propranolol-
pretreated rats was significantly lower than that in
microsomes from rats receiving saline only and was
comparable to that from male DA rats. The effect of the
P4502D inhibitor, debrisoquine, on the formation of PTP
was also examined. The results, shown in Figure 5,
activity in liver microsomes from male Wistar rats. The
activity was almost completely (over 99%) inhibited by
SKF 525-A (2 mM, data not shown). Omission of
NADPH, the cofactor for monooxygenation by P450,
prevented the formation of PTP. These results demon-
strated that MPTP N-demethylation was mediated by
P450 enzyme in rat liver microsomes under the present
conditions.
Kin etics of MP TP N-Dem eth yla tion . We then
examined the kinetics of formation of PTP from MPTP
in liver microsomes from male Wistar rats using a
substrate concentration range from 1.25 to 5000 µM. The
results are shown in Figure 3 as a nonlinear Eadie-
Hofstee plot which indicates multiple enzyme participa-
tion. The data were analyzed in terms of a two enzyme
system by nonlinear regression procedures, and the
+
+

9
6
Chem. Res. Toxicol., Vol. 9, No. 1, 1996
Narimatsu et al.
Ta ble 1. MP TP N-Dem eth yla se Activity of P u r ified P 450
En zym es in th e Recon stitu ted System a
MPTP N-demethylase act.
(
nmol/(min‚nmol of P450))
at a substrate concn of:
enzymes
5 µM
5 mM
P450BTL
P450m1
0.19
0.57
0.23
101.8
a
The reconstituted system (0.5 mL) contained purified P450
50 pmol), NADPH-P450 reductase (0.5 unit), sodium cholate (0.1
(
mg), dilauroylphosphatidylcholine (5 µg), NADPH (0.5 µmol),
MPTP (2.5 nmol or 2.5 µmol), the NADPH-generating system, and
potassium phoshate buffer (pH 7.4, 77 µmol). Each value repre-
sents the mean of two determinations.
F igu r e 4. Effects of pretreatment of rats with P450 inducers
on and strain and sex differences in MPTP N-demethylase
activities in rat liver microsomes. For the assessment of the
effects of inducers, male Wistar rats were given a daily dose of
phenobarbital, â-naphthoflavone (each 80 mg/kg, ip, for 3 days),
or propranolol (100 mg/kg, po, for 5 days) and were sacrificed
2
4 h (phenobarbital and â-naphthoflavone) or 40 h (propranolol)
after the final dose. For the assessment of sex difference, liver
microsomes were prepared from Wistar and DA rats of both
sexes. MPTP N-demethylase activities were assayed using
substrate concentrations of 5 µM (A and C) and 5 mM (B). Each
value represents the mean (SE of three animals. WM, DAM,
and DAF mean Wistar male, Wistar female, DA male, and DA
female, respectively. WM+PB, WM+BNF, and WM+PL mean
phenobarbital-, â-naphthoflavone-, and propranolol-treated male
Wistar rats, respectively. (*, **) Significantly different from
male Wistar rats (p <0.05 and p <0.01, respectively). (§, §§)
significantly different from male DA rats (p <0.05 and p <0.01,
respectively). (##) Significantly different from female Wistar
rats (p <0.01).
F igu r e 6. Effects of antibodies against P450BTL and P450m1
on MPTP N-demethylase activities in rat liver microsomes.
Polyclonal antibodies against P450BTL or P450m1 were pre-
incubated with liver microsomes from male Wistar rats at 25
°C for 30 min. MPTP and an NADPH-generating system were
then added, and incubation at 37 °C was performed. Closed
circles, anti-P450 IgG; open circles, preimmune IgG. Left panels
(A and C), low substrate concentration (5 µM); right panels (B
and D), high substrate concentration (5 mM); upper panels (A
and B), anti-P450BTL; lower panels (C and D), anti-P450m1.
Each point was the mean of duplicated experiments.
of the activity remained even at the highest debrisoquine
concentration of 500 µM.
Recon stitu tion of MP TP N-Dem eth yla tion . MPTP
N-demethylase activity was then reconstituted, using
purified P450 enzymes (P450BTL and P450m1), NADPH-
P450 reductase, dilauroylphosphatidylcholine, and an
NADPH-generating system (Table 1). P450m1 showed
PTP-forming activity higher than that of P450BTL at
both substrate concentrations of 5 µM and 5 mM. At 5
mM, the activity of P450m1 was 101.8 nmol/(min‚nmol
of P450), which was 446-fold higher than that of P450BTL.
Effects of An tibod ies a ga in st P 450 En zym es on
Micr osom a l MP TP N-Dem et h yla t ion . Liver mi-
crosomes from male Wistar rats were preincubated with
antibodies raised against P450BTL or P450m1, and
microsomal MPTP N-demethylase activity was assayed
using the two substrate concentrations. As shown in
Figure 6, antibodies against P450BTL suppressed the
formation of PTP from MPTP at a low substrate concen-
tration (5 µM) by 48% at an antibody to microsomal
protein ratio of 5 (Figure 6A), whereas antibodies against
F igu r e 5. Effects of debrisoquine on MPTP N-demethylase
activities in rat liver microsomes. Debrisoquine (final concen-
trations of 50-500 µM) was added to the incubation medium
containing microsomes from male Wistar rats and other ingre-
dients and was incubated at 37 °C for 2.5 min. Each value
represents the mean (SE of three determinations.
indicate that debrisoquine suppressed MPTP N-dem-
ethylase activity in a concentration-dependent manner.
However, the suppression was incomplete, and about 40%
+
+

MPTP N-Demethylation by Rat Liver P450 Enzymes
Chem. Res. Toxicol., Vol. 9, No. 1, 1996 97
P450m1 suppressed the activity only by 22% even at the
highest protein ratio (Figure 6C). When the high sub-
strate concentration (5 mM) was employed, on the other
hand, the antibodies against P450BTL had no effect on
the activity (Figure 6B), whereas the antibodies against
P450m1 reduced MPTP N-demethylation activity to 22%
of control at an antibody to microsome protein ratio of
nmol/(min‚nmol of P450). Moreover, the immunochemi-
cal studies demonstrated that antibodies against P450BTL
suppressed half of the activity at the low MPTP concen-
tration, whereas antibodies against P450m1 suppressed
most of the activity at the high MPTP concentration.
These results indicate that major P450 enzymes cata-
lyzing MPTP N-demethylation are P4502D enzyme(s) as
1
.0 (Figure 6D).
a low K
and P4502C11 as a high K
m
enzyme in a lower substrate concentration range
enzyme in a higher substrate
m
concentration range in rat liver microsomes.
Discu ssion
In the present study, MPTP N-demethylation in liver
microsomes from male Wistar rats was found to be
biphasic, i.e., consistent with two enzyme systems. This
means at least two P450 enzymes are involved in the
formation of PTP from MPTP in male rat liver mi-
crosomes. Chiba et al. (8) reported the kinetics of PTP
from MPTP in mouse liver microsomes was monophasic,
The present study showed that MPTP N-demethylase
activity was lower in DA rats than in Wistar at a lower
substrate concentration. Furthermore, a male-specific
P4502C11 is supposed to contribute to PTP formation at
higher substrate concentrations. These results and find-
ings suggest that lower MPTP N-demethylase activity
makes female DA rats more sensitive to neurotoxicity of
MPTP than male rats of other strains. In fact, J imenez-
J imenez et al., (29) reported that female DA rats are more
susceptible to acute neurotoxic effects of MPTP than
other strain rats such as Fischer-344 or Wistar. How-
ever, there have been no reliable reports showing a clear
sex difference in MPTP neurotoxicity in any animal
species including the rat.
with a K
of 4.42 ( 0.28 nmol/(min‚mg of protein). These param-
eter values are similar to those for the high K system
m2, 1180 ( 91 µM; Vmax2, 4.80 ( 0.75 nmol/(min‚mg of
protein)) in rat liver microsomes in our study.
m
value of 814 ( 71 µM and a maximum velocity
m
(
K
They also observed that the pretreatment of mice with
phenobarbital or 3-methylcholanthrene increased the
clearance (8), suggesting the involvement of P450 en-
zymes belonging to the P4502B or -1A subfamilies in
mouse liver microsomal MPTP N-demethylation. Con-
trary to the results in the mouse (8), however, pretreat-
ments of rats with P450 inducers such as phenobarbital
and â-naphthoflavone markedly decreased the microsome
mediated PTP formation, so it is unlikely that P450
enzymes belonging to the P4501A or -2B subfamily are
involved in MPTP N-demethylation by this species.
MPTP N-demethylase activity was significantly lower
in the P4502D deficient DA rat (27) than in Wistar rats,
particularly at a low substrate concentration (5 µM),
indicating that P4502D enzyme(s) were involved in rat
liver microsomal MPTP N-demethylation. Previous stud-
ies (26) had shown that propranolol selectivity inhibits
P4502D enzymes. We thus examined MPTP N-demethy-
lase activities in liver microsomes from rats pretreated
with propranolol and found that the activity of male
Wistar rats receiving the propranolol pretreatment was
decreased to the level of native male DA rats, whose
activity was significantly lower than that of native male
Wistar rats. These results indicated that P4502D enzyme-
We reported previously that 1,2,3,4-tetrahydroiso-
quinoline, another compound that induces a Parkinso-
nism-like state in experimental animals, was converted
to a nontoxic 4-hydroxylated metabolite mainly by P4502D
enzyme(s) in the rat (30). The results of the present
study show that metabolism of MPTP by P4502D enzyme-
(s) and P4502C11 is a detoxication pathway in male rat
liver. It is known that mammalian brains contain
various P450 enzymes including the P4502D subfamily
(31, 32). These results and findings do not directly
support the possible correlation between the debrisoquine
hydroxylase deficient poor metabolizer genotype and
Parkinsonism (17-19, 33), but instead suggest that
P4502D enzymes are involved in the detoxication of these
neurotoxic substances in mammalian brains as well as
in livers. It is tempting to think that, in the brain, some
interaction between P4502D enzyme(s) and neurotoxins
such as MPTP or 1,2,3,4-tetrahydroisoquinoline in the
situation of enzyme-substrate or enzyme-inhibitor would
be one of causes correlating P4502D polymorphism with
Parkinson’s disease, but supporting data are still insuf-
ficient. Further systematic studies focusing on this point
are needed.
(
s) are involved in MPTP N-demethylation in a low
substrate concentration range.
Ack n ow led gm en t. We express our gratitude to Mr.
Kenji Matsuura, J EOL, Shojima, Tokyo, for performing
the LC/MS analysis of the MPTP metabolites.
We observed a marked sex difference (male > female)
in MPTP N-demethylase activities in liver microsomes
from Wistar and DA rats, especially at a high substrate
concentration. It should be noted that there was no
strain difference between Wistar and DA rats in MPTP
N-demethylase activities at a MPTP concentration of 5
mM (Figure 4B). These results indicate that male-
specific P450 enzyme(s) are the major contributors to PTP
formation in male rat liver microsomes as the major P450
at higher substrate concentration range.
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