Neuroprotection in the MPTP parkinsonian C57BL/6 mouse model by a compound isolated from tobacco

Article abstract of DOI:10.1021/tx000224v

Epidemiological evidence suggests a lower incidence of Parkinson's disease in smokers than in nonsmokers. This evidence, together with the lower levels of brain monoamine oxidase (MAO) activity in smokers and the potential neuroprotective properties of MAO inhibitors, prompted studies which led to the isolation and characterization of 2,3,6-trimethyl-1,4-naphthoquinone (TMN), an MAO-A and MAO-B inhibitor which is present in tobacco and tobacco smoke. Results of experiments reported here provide evidence that this compound protects against the MPTP-mediated depletion of neostriatal dopamine levels in the C57BL/6 mouse. These results support the hypothesis that the inhibition of MAO by constituents of tobacco smoke may be related to the decreased incidence of Parkinson's disease in smokers.

Full text of DOI:10.1021/tx000224v

Chem. Res. Toxicol. 2001, 14, 523-527
523
Neu r op r otection in th e MP TP P a r k in son ia n C57BL/6
†
Mou se Mod el by a Com p ou n d Isola ted fr om Toba cco
,
‡
‡
‡
Kay P. Castagnoli,* Stefanus J . Steyn, J acobus P. Petzer,
Cornelis J . Van der Schyf,‡ and Neal Castagnoli, J r.‡
,§
Harvey W. Peters Center, Department of Chemistry, and Department of Biomedical Sciences and
Pathobiology, Virginia Tech, Blacksburg, Virginia 24061-0212
Received October 23, 2000
Epidemiological evidence suggests a lower incidence of Parkinson’s disease in smokers than
in nonsmokers. This evidence, together with the lower levels of brain monoamine oxidase (MAO)
activity in smokers and the potential neuroprotective properties of MAO inhibitors, prompted
studies which led to the isolation and characterization of 2,3,6-trimethyl-1,4-naphthoquinone
(
TMN), an MAO-A and MAO-B inhibitor which is present in tobacco and tobacco smoke. Results
of experiments reported here provide evidence that this compound protects against the MPTP-
mediated depletion of neostriatal dopamine levels in the C57BL/6 mouse. These results support
the hypothesis that the inhibition of MAO by constituents of tobacco smoke may be related to
the decreased incidence of Parkinson’s disease in smokers.
In tr od u ction
1
Parkinson’s disease (PD) is an age-dependent, neuro-
degenerative disorder involving the selective loss of
dopaminergic nigrostriatal neurons (1). Since no strong
genetic link has been identified for this disease, other
factors, including exposure to environmental neurotoxins,
have been proposed to play a critical role in its etiology
(
2). The characterization of the parkinsonian-inducing
properties of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyri-
dine [MPTP (1); see Figure 1 for structures] more than
1
5 years ago (3) has lent additional support to the
environmental neurotoxin theory and also has led to the
development of animal models for studying how such
neurotoxins might lead to pathological lesions of the
nigrostriatal system. A useful animal model for studying
neurodegenerative and neuroprotective processes is the
MPTP-treated C57BL/6 mouse (4, 5). In one variation of
this model, a single intraperitoneal (ip) dose of MPTP
leads to the loss of nigrostriatal dopaminergic cell bodies
in the substantia nigra and depletion of neostriatal
dopamine (DA) levels (6-8).
The molecular mechanism by which MPTP selectively
damages nigrostriatal neurons has been the subject of
extensive research, and much is known (9). Critical to
its mode of action is the monoamine oxidase B (MAO-
B)-catalyzed allylic R-carbon oxidation of the parent
F igu r e 1. Structures of compounds discussed in the text.
tetrahydropyridine, leading to the corresponding dihy-
+
dropyridinium species MPDP (2) (10). This intermediate
undergoes a second two-electron oxidation to generate
+
the pyridinium metabolite MPP (3), the ultimate neu-
rotoxin (10).
Several compounds have been reported to protect
against MPTP’s nigrostriatal toxicity in the C57BL/6
mouse. The mechanisms by which most of these com-
pounds act are only poorly understood [for a recent
review, see Alexi et al. (11)]. One well-documented
neuroprotective pathway involves inhibition of the MAO-
†
A preliminary report of this work was presented at the American
Chemical Society Meeting, San Francisco, CA (March 2000), and the
Millennium Monoamine Oxidase Meeting, Barcelona, Spain (J uly
+
B-catalyzed bioactivation of MPTP to MPP . For ex-
2
000).
To whom correspondence should be addressed: Department of
Chemistry, Virginia Tech, Blacksburg, VA 24061-0212. Telephone:
*
ample, pretreatment of C57BL/6 mice with (R)-deprenyl
(4), or other potent and selective mechanism-based
(
540) 231-8200. Fax: (540) 231-8890. E-mail: kcastagn@vt.edu.
‡
§
1
inactivators of MAO-B, renders animals resistant to the
neurotoxic effects of MPTP (12-16). Also of interest is
the use of this MAO-B inhibitor to treat patients diag-
nosed with PD (17).
One of the interesting outcomes of epidemiological
studies on PD is the clear evidence of a lower incidence
of this disease in tobacco smokers than in nonsmokers
Harvey W. Peters Center, Department of Chemistry.
Department of Biomedical Sciences and Pathobiology.
Abbreviations: MAO, monoamine oxidase; GC/EIMS, gas chro-
matography/electron ionization mass spectrometry; PD, Parkinson’s
disease; TCA, trichloroacetic acid; MPTP, 1-methyl-4-phenyl-1,2,3,6-
+
tetrahydropyridine; MPDP , 1-methyl-4-phenyl-2,3-dihydropyridinium
+
ion; MPP , 1-methyl-4-phenylpyridinium ion; TMN, 2,3,6-trimethyl-
1
,4-naphthoquinone; 7-NI, 7-nitroindazole; DA, dopamine; MP, mobile
phase; IS, internal standard.
1
0.1021/tx000224v CCC: $20.00 © 2001 American Chemical Society
Published on Web 04/06/2001

5
24 Chem. Res. Toxicol., Vol. 14, No. 5, 2001
Castagnoli et al.
(
18). Consistent with this antiparkinsonian effect is the
of the synthetic intermediate 2,3-dimethyl-1,4-benzoquinone (7)
which was synthesized as follows. A solution of 2,3-dimeth-
ylphenol (3.1 g, 25 mmol) and hydrated salcomine (1.6 g, 5
mmol) (36) in 100 mL of N,N-dimethylformamide was stirred
under an oxygen atmosphere for 24 h. After addition of an equal
volume of brine, the solution was filtered, the filtrate was
extracted with diethyl ether (3 × 300 mL), and the combined
organic layers were filtered. The filtrate was washed twice with
report by Carr et al. (19) that tobacco smoke attenuates
the neurotoxicity of the parkinsonian-inducing drug
MPTP in the C57BL/6 mouse model. These results have
prompted us to consider the possibility that tobacco and/
or tobacco smoke may contain one or more neuroprotec-
tive compounds that act via inhibition of MAO. Early
studies by Essman (20) showed that in mouse skin
exposed to cigarette smoke, the level of MAO-catalyzed
oxidation of serotonin was decreased. Norman et al. (21)
claimed that cigarette smoke inhibited MAO activity.
Carr et al. (22) in mouse brain homogenates and Yu et
al. (23) in rat lung tissue have shown MAO inhibition
with tobacco smoke extracts, although nicotine itself, at
physiologically significant levels, was shown not to
contribute to this inhibition (22). In addition, a number
of authors have shown that the human blood platelet
MAO activity (MAO-B) is significantly reduced in smok-
ers compared to that in nonsmokers (24-26). Even more
relevant are the recent reports by Fowler et al. (27, 28)
using in vivo positron emission tomography that dem-
onstrated lower MAO-A and MAO-B activities in the
brains of smokers.
Recent efforts in our laboratory have led to the isola-
tion from tobacco leaf extracts of 2,3,6-trimethyl-1,4-
naphthoquinone [TMN (5)], a compound which competi-
tively inhibits both MAO-A (K
)
tobacco leaves and tobacco smoke (30, 31). In this paper,
we describe the results of studies aimed at assessing the
potential in vivo inhibition of brain MAO by TMN and
its neuroprotective properties in the C57 parkinsonian
mouse model.
2 4
an equal volume of brine, dried over Mg SO , and concentrated
to a small volume which was purified by passage through a
column of neutral, activated alumina (50 g) using 9:1 hexane/
ethyl acetate. Fractions (5 mL) containing the desired product
(3-25) were combined and evaporated to dryness in vacuo, and
the residue was recrystallized from n-hexane to give the pure
product (1.1 g, 33%): mp 55 °C [lit. mp 56-57 °C (37)].
HP LC Assa y for TMN. TMN analyses were performed on a
Hewlett-Packard 1100 HPLC system equipped with a UV/vis
diode array (DA) detector, a Rheodyne 7725I injector, an Altech
adsorbosphere C18 analytical column (4.6 mm × 150 mm, 5 µm)
preceded by a C18 guard column (Opti Guard, 1 mm; Altech).
The mobile phase (MP) consisted of 70% methanol and 30%
Milli-Q water with a flow rate of 1 mL/min. The IS (6) and TMN
were monitored at 265 nm. Calibration curves for the quantita-
tive measurements of TMN were constructed using standards
prepared in 65% MeOH and 35% Milli-Q water, containing 5%
TCA (w:v) (solution A) and 1.3 µM IS with concentrations of
TMN ranging from 0.5 to 9.0 µM. These calibration standards
were injected (200 µL) into the HPLC system, and the TMN:IS
peak height ratios were plotted versus the micromolar TMN
concentration.
I
) 3 µM) and MAO-B (K
I
6 µM) (29) and which has been shown to be present in
Protocols for all animal experiments were reviewed and
approved by the Virginia Tech Animal Care Committee and
follow the Guide for the Care and Use of Laboratory Animals,
7th ed., 1996, National Research Council.
TMN R ecover y fr om Br a in Hom ogen a t es. Two sets of
samples were utilized to measure the recovery of TMN from
mouse brain homogenates. Brain tissues were homogenized in
solution A (6 µL/mg of wet tissue). Each sample for group 1 (n
Exp er im en ta l P r oced u r es
)
3) consisted of 343 µL of brain homogenate, 100 ng of internal
Ca u tion : MPTP is a known nigrostriatal neurotoxin and
should be handled following established procedures (32).
Ch em ica ls, Rea gen ts, a n d Gen er a l Meth od s. Chemicals
and reagents not described elsewhere were purchased from
Aldrich Chemical Co. (Milwaukee, WI). Milli-Q water was
obtained from a Waters Milli-Q UV Plus system (Millipore,
Bedford, MA). The following were obtained as indicated: HPLC
grade methanol (Burdick & J ackson, Muskegon, MI); trichlo-
standard in 10 µL of solution A, 280 ng of TMN in 28 µL of
solution A, and an additional 19 µL of solution A to give a final
volume of 400 µL. For group 2 (n ) 3), essentially the same
procedure was followed except that 28 µL was added to the
homogenate instead of the TMN. Each sample was again
homogenized and then centrifuged for 10 min at 15000g. An
aliquot of each supernatant (300 µL) was transferred to a new
microcentrifuge tube. Solution A (21 µL) was added to samples
in group 1, and TMN (210 ng in 21 µL of solution A) was added
to group 2 samples which were then vortexed. An aliquot (200
µL) was injected into the HPLC system. Recoveries were
determined by comparing the TMN:IS peak height ratios from
group 1 samples to those from group 2.
roacetic acid (TCA), 50% H
2 2 2 4
O , enzyme grade Na HPO and
NaH PO , Na SO , and glycerol (Fisher Scientific Products, Fair
2
4
2
4
Lawn, NJ ); Hollywood peanut oil (Kroger Inc., Blacksburg, VA);
dopamine hydrochloride (DA) and 3,4-dihydroxybenzylamine
hydrobromide (Research Biochemicals Inc., Natick, MA); and
hydrated salcomine (Sigma Chemical Co., St. Louis, MO). TMN
was synthesized as reported previously (29) with minor modi-
fications as described below. MPTP‚HCl (33) was synthesized
according to literature methods. Melting points were obtained
on a Thomas-Hoover melting point apparatus and are uncor-
rected. NMR spectra were obtained on a Brucker WP 360 MHz
spectrometer. The GC/MS system was as follows: Hewlett-
Packard MSD 5870, GC 5890, ChemStation 5970, HP-1 column,
Bioa va ila bility of TMN in th e Br a in . Injection solutions
of TMN were prepared in peanut oil by heating the mixture for
20 min at 50 °C. Male C57BL/6 mice, 11-13 months of age
(Harlan-Sprague Dawley, Dublin, VA), were treated sc with
TMN at doses ranging from 200 to 400 mg/kg of body weight in
0.2-0.3 mL. Animals were sacrificed at time points postinjection
ranging from 10 to 120 min. Each brain was removed, weighed,
and homogenized in 6 µL/mg of the IS solution (1.34 µM in
solution A). The resulting homogenate was centrifuged for 10
min at 15000g. The supernatant was transferred to a clean tube
and recentrifuged for 5 min at 15000g. The resulting superna-
tant was analyzed by the HPLC assay described previously.
2
5 m × 0.2 mm × 0.33 µm, split/splitless injector, splitless mode,
port temperature of 250 °C; GC oven program, from 60 to 290
°
C at a rate of 25 °C/min.
Syn t h eses. (1) 2,6-Dim et h yl-1,4-n a p h t h oq u in on e (6).
This compound, which was used as an internal standard (IS)
in the assay developed to estimate brain concentrations of TMN,
was synthesized following the literature procedure for the
synthesis of 2-methyl-1,4-naphthoquinone (34) using commer-
cially available 2,6-dimethylnaphthalene as a starting mate-
rial: mp 133-136 °C [lit. mp 134-135 °C (35)].
Neu r op r otection Stu d ies in th e C57BL/6 Mou se. Eleven-
month-old, retired male breeder C57BL/6 mice (Harlan-Sprague
Dawley) were housed one per cage in a temperature-controlled
room with free access to food and water on a 12 h light/dark
cycle. Injection solutions of TMN (189 mM) in peanut oil were
prepared by stirring and heating at 50 °C for 20 min. Control
mice (n ) 4) were treated sc with peanut oil (0.32 mL/30 g of
body weight) 25 min prior to the administration of sterile saline
(
2) 2,3,6-Tr im eth yl-1,4-n a p h th oqu in on e [TMN (5)] was
prepared as described previously (29) except for the preparation

Tobacco Constituents and Neuroprotection
0.2 mL ip). MPTP-treated mice (n ) 10) were treated sc with
Chem. Res. Toxicol., Vol. 14, No. 5, 2001 525
(
peanut oil (0.32 mL/30 g) 25 min prior to MPTP (35 mg/kg of
MPTP‚HCl) ip administration. Mice treated with TMN and
MPTP (n ) 7) were administered TMN (400 mg/kg) 25 min prior
to MPTP‚HCl ip administration (35 mg/kg). The animals were
sacrificed 10 days after drug administration, and the striata
were dissected free; 10 µL of the IS solution {3,4-dihydroxyben-
zylamine [6.82 µM in 5% TCA (w:v) in Milli-Q water]} was added
per milligram of wet striatal tissue, and the striata were
homogenized. The samples were subsequently centrifuged
(16000g), and 20 µL aliquots of the resulting supernatants were
analyzed for DA by HPLC as previously described (37, 38) using
a Bioanalytical C4 system with a PM 60 pump (BAS, India-
napolis, IN) and a Kipp and Zonen BD 41 recorder.
Resu lts a n d Discu ssion
In addition to the well-characterized effects of irrevers-
ible MAO-B inhibitors in the C57 mouse PD model,
reversible inhibitors of MAO-B also have been shown to
protect against the neurotoxic effects of MPTP in this
model by inhibiting the MAO-B-catalyzed bioactivation
of this proneurotoxin (40-42). Our experience is with
7
-nitroindazole [7-NI (8)], a selective inhibitor of neuronal
nitric oxide synthase (43) that also is a competitive
inhibitor (K
I
) 4 µM, mouse brain mitochondria) of
MAO-B (44). Administration of this compound to the
C57BL/6 mouse prior to MPTP decreased the extent of
conversion of MPTP to MPP in the brain and protected
+
F igu r e 2. Striatal dopamine levels ( SEM (picomoles per
milligram of wet tissue) in C57BL/6 male retired breeder mice
in the following treatment groups: control (n ) 4), no drug
treatment; MPTP (n ) 10), peanut oil sc 25 min prior to MPTP‚
HCl in sterile saline ip at 35 mg/kg; TMN + MPTP (n ) 7),
TMN in peanut oil at 400 mg/kg sc 25 min prior to MPTP‚HCl
in sterile saline ip at 35 mg/kg.
the mice against the MPTP-induced long-lasting deple-
tion of neostriatal DA levels (6, 7, 44). Using these studies
as a guide, we proceeded to examine the neuroprotective
properties of TMN which also would provide an indication
of its in vivo MAO-B inhibiting properties.
The poor water solubility of TMN led us to administer
the compound sc in peanut oil, a procedure which has
been used by us and others with 7-NI (6, 45). Estimates
of the extent to which TMN distributes to the brain under
these conditions were made employing an HPLC diode
array assay using 2,6-dimethyl-1,4-naphthoquinone (6)
it rapidly declines (44). Consequently, MPTP was ad-
ministered 25 min after a sc 400 mg/kg dose of TMN.
Since the K value for the MAO-B-catalyzed oxidation
m
of MPTP is 40 µM in mouse brain mitochondrial prepara-
tions (44), we anticipated that the bioactivation reaction
would be significantly inhibited by the TMN concentra-
tion of 40 µM achieved under these conditions. Similar
brain concentrations of 7-NI (20 µmol/kg), which has a
as the IS. Calibration curves with good linearity (r2
.991) and coefficients of variation (n ) 3) between 5.5
and 6.5% were obtained over a concentration range of
.5-9.0 µM TMN, while recoveries of TMN-spiked whole
)
0
K value of 4 µM for this reaction, were effective in
I
0
inhibiting the MAO-B-catalyzed oxidation of MPTP‚HCl
brain homogenates were found to be 73.7 ( 2.0% (SD)
(0.24 mmol/kg, equivalent to 50 mg/kg).
(see Experimental Procedures).
Three groups of mice were used for the neuroprotection
Utilizing this assay, we measured the TMN brain
experiments. Control mice (n ) 4) received no drug
treatment; MPTP mice (n ) 10) received peanut oil sc
concentrations of mice treated sc with various doses of
TMN in peanut oil (1-2 mmol/kg) and at varying
postinjection times (10-120 min). The results established
that the brain concentrations are maximal 25 min
postinjection. Unfortunately, only 0.003-0.023% of the
doses that were administered partitioned into the brain.
Part of the problem appeared to be due to the tendency
of these oily preparations to form saclike structures at
the sites of injection. This behavior was not observed with
2
5 min prior to MPTP‚HCl administration (ip 0.17 mmol/
kg ) 35 mg/kg in sterile saline), and TMN mice (n ) 7)
received TMN (sc 400 mg/kg in peanut oil) followed 25
min later by MPTP (ip 0.17 mmol/kg in sterile saline).
The animals were sacrificed 10 days later; the striata
were isolated, and the concentrations of DA were deter-
mined using HPLC with electrochemical detection (38,
3
(
2
9). The control mice had neostriatal DA levels of 138.54
3.26 (SEM) pmol/mg of wet tissue (100.0%) (see Figure
). The corresponding DA levels in mice that received
only MPTP were 57.01 ( 8.67 pmol/mg of wet tissue or
41.1% of the control levels. The DA levels in the animals
7
6
-NI. Brain concentrations of ∼40 µmol TMN/kg, or about
.6 times the K value for the inhibition of MAO-B, were
I
achieved 25 min postinjection when a dose of 400 mg/kg
was administered with gentle massaging at the site of
injection.
In the neuroprotection studies, we chose an ip dose of
MPTP‚HCl (0.17 mmol/kg, equivalent to 35 mg/kg ip)
which, in our experience, leads to an ∼40% decrease in
the concentration of DA in the neostriata of C57BL/6
mice. When administered ip, MPTP reaches its maximum
brain concentration at approximately 10 min and then
∼
treated with TMN and MPTP were 82.96 ( 6.83 pmol/
mg of wet tissue or 59.9% of control levels. These striatal
DA concentrations in both groups treated with MPTP are
significantly different from those in control animals (p
< 0.0001 for control vs MPTP only, p < 0.0003 for control
vs TMN and MPTP, Student’s t test, Instat for Graphics
4). However, the mice treated with TMN and MPTP

5
26 Chem. Res. Toxicol., Vol. 14, No. 5, 2001
Castagnoli et al.
exhibited an increase in DA content, compared to animals
treated with only MPTP, of ∼50%, and a comparison of
these values demonstrates significant difference (p <
(3) Langston, J . W., Ballard, P., Tetrud, J . W., and Irwin, I. (1983)
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analog synthesis. Science 219, 979-980.
(
(
(
4) Heikkila, R. E., Hess, A., and Duvoisin, R. C. (1984) Dopaminergic
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M., Ed.) pp 142-143, Humana Press, Totowa, NJ .
0
.05). These results are quite similar to those obtained
when 7-NI was administered prior to MPTP. In those
earlier studies, 7-NI pretreatment (sc 50 mg/kg in peanut
oil) 25 min prior to MPTP‚HCl administration (0.194
mmol/kg ) 40 mg/kg) also led to an increase of ∼50% in
the striatal DA concentrations compared to mice treated
with MPTP only. It should also be mentioned that the
mice pretreated with TMN appeared to be sluggish prior
to treatment with MPTP, while the mice pretreated with
vehicle (peanut oil) did not exhibit this behavior. Both
groups displayed the early onset characteristics of MPTP
treatment, i.e., shakiness, Straub tail, and some difficulty
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(
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These studies establish that, under the experimental
conditions that were utilized, the tobacco constituent
TMN, which we have previously shown to be a reversible
inhibitor of human MAO-A and MAO-B, is protective in
the C57BL/6 mouse against the neurotoxicity of MPTP.
The poor and variable partitioning characteristics of
TMN when administered sc in peanut oil required a very
high dose of the drug to achieve the brain concentrations
observed in these studies. What brain levels might be
achieved in smokers, who will be self-dosing with TMN
on a continuous basis, is hard to predict. Nevertheless,
it may be reasonable to speculate that the well-docu-
mented lowered activity of MAO in the brain of smokers
may be in part mediated by TMN. Also noteworthy is
recent evidence from our laboratory that more than one
inhibitor of MAO may be present in the tobacco plant,
since significant MAO-B inhibiting activity has been
observed in tobacco leaf extracts with widely different
chromatographic polarities compared to TMN, and we
have evidence that both reversible and irreversible
inhibitors of MAO-A and MAO-B are present in tobacco
smoke hexane extracts.
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attenuated brain MAO activity levels in human smokers
and the lowered incidence of PD have been established,
these findings, together with results of studies on tobacco
smoke exposure itself, provide evidence that constituents
of tobacco can provide neuroprotection in a well-charac-
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clearly lowered incidence of PD in human smokers and
a possible relationship to components in the tobacco leaf
and tobacco smoke which may include reversible and
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(
Ack n ow led gm en t. We thank Mr. David Gemmell
and the staff of the Laboratory Animal Resources facility
for their support. This work was supported by a grant
from the National Institute on Drug Abuse (DA11089),
a gift from Reverend Stewart Bryan West, and the
Harvey W. Peters Center for the Study of Parkinson’s
Disease, Department of Chemistry, Virginia Tech.
(
(
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