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Research report
Increased striatal dopamine turnover following acute administration of
rotenone to mice
*
Christine Thiffault , J. William Langston,, Donato A. Di Monte
The Parkinson’s Institute, Sunnyvale, CA 94089, USA Accepted 12 September 2000
Abstract
Because of the potential role of mitochondrial dysfunction in nigrostriatal degeneration in Parkinson’s disease, the effects of rotenone (an inhibitor of mitochondrial NADH dehydrogenase and a naturally occurring toxicant) on the levels of striatal dopamine (DA) and DA metabolites were evaluated after acute and subchronic administration to mice. Systemic acute treatment with relatively high doses of rotenone did not affect DA concentration, but caused a significant increase in both DA metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA). DOPAC and HVA changes were measured at 1 day and were reversed within 1 week, paralleling the time course of rotenone-induced increase in striatal lactate levels. Subchronic administration with a relatively mild dose of rotenone did not significantly alter the striatal levels of DA and DOPAC, while it slightly reduced HVA concentration. No neurochemical signs of dopaminergic damage were seen when mice were co-exposed to rotenone and diethyldithiocarbamate, a compound known to enhance nigrostriatal injury caused by the neurotoxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Also, rotenone did not cause additional injury to animals previously lesioned by MPTP. Taken together, data indicate that rotenone is not capable of causing overt dopaminergic toxicity under the testing paradigms used in this study. Rather, an increase in DA turnover, as indicated by a higher (DOPAC1HVA) / DA ratio, seems to be associated to rotenone-induced striatal energy impairment. 2000 Elsevier Science B.V. All rights reserved.
Theme: Disorders of the nervous system
Topic: Neurotoxicity
Keywords: NADH dehydrogenase; Rotenone; Mitochondrial respiratory dysfunction; Diethyldithiocarbamate; MPTP; Dopamine metabolism.
1
1. Introduction metabolite (MPP ), involves the inhibition of complex I activity of the mitochondrial respiratory chain [23–25],
The neurotoxic effects of 1-methyl-4-phenyl-1,2,3,6- with consequent ATP depletion and a loss of mitochondrial
tetrahydropyridine (MPTP) reproduce neurochemical and transmembrane potential [3,5,12,14,16,28]. Similar toxic
pathological features of human parkinsonism such as the events may play a role in neurodegeneration in PD since a
reduction of striatal dopamine (DA) and the loss of decrease in complex I activity has been measured post
dopaminergic cell bodies in the substantia nigra (SN) mortem in both the SN and the striatum of PD patients as
[3,14,17,29]. Thus, studies on MPTP toxicity are likely to compared to control subjects [22,27].
provide valuable insight into mechanisms of nigrostriatal Findings with the MPTP model and in PD patients raise
degeneration in Parkinson’s disease (PD). Several lines of the possibility that the nigrostriatal system may be
par-experimental evidence suggest that MPTP-induced cell ticularly vulnerable to an impairment of mitochondrial
death, mediated by its toxic 1-methyl-4-phenylpyridinium energy metabolism. Rotenone is a ‘classic’ inhibitor of
mitochondrial complex I and could therefore be used to further evaluate the relationship between energy deficiency
*Corresponding author. Department of Neurology, Health Science
and dopaminergic injury. Furthermore, since rotenoid
Center, Box 800394, University of Virginia, Charlottesville, VA 22908,
compounds are widely used as pesticides, studies with
USA. Tel.:11-804-924-8374; fax:11-804-982-1276.
E-mail address: [email protected] (C. Thiffault). rotenone may provide clues on environmental exposures
that could increase the risk for PD. Particularly intriguing Guide for the Care and Use of Laboratory Animals and
is the possibility that chemical interactions may play a role were approved by the Institutional Animal Care and Use
in nigrostriatal degeneration. The fungicide diethyldithio- Committee. Mice received a single subcutaneous injection
carbamate (DDC) has been shown to enhance dramatically of rotenone (5, 10 and 15 mg / kg) or vehicle (peanut oil)
MPTP-induced neurotoxicity [6,13,31]. However, whether and were killed by cervical dislocation at 1, 7 or 14 days.
DDC can also act synergistically with other compounds, With the highest dose of rotenone chosen for these
including rotenoid derivatives, in damaging dopaminergic experiments, i.e. 15 mg / kg, mice survived 24 h, but
neurons is unknown. A limited number of studies has significant lethality was observed at subsequent time
evaluated the effects of rotenone on dopaminergic neurons points. Therefore, this dose was used only to determine the
both in vitro and in vivo. Treatment of mesencephalic most severe acute effects of rotenone at 1 day. For the
cultures and striatal synaptosomes with rotenone has been subchronic regimen, mice were injected with 1 / 10 of the
shown to cause neurotoxicity as measured by a decreased highest acute dose of rotenone (1.5 mg / kg, s.c.) three
uptake of neurotransmitters [20,21]. Interestingly, DA times a week (Monday, Wednesday and Friday) for 3
uptake was significantly more affected than the uptake of consecutive weeks and were killed on the third day after
serotonin, GABA or noradrenaline, thus supporting the the last dose. In studies in which chemical interactions
hypothesis of a selective vulnerability of dopaminergic were evaluated, DDC (400 mg / kg, i.p.) was administered
cells. In the in vivo setting, Heikkila and colleagues have 2 h before rotenone (10 mg / kg, s.c.) or vehicle and mice
reported a substantial depletion of striatal DA and its were killed at 7 days. In a final set of experiments, mice
metabolites after the stereotaxic injection of rotenone into received MPTP (15 mg / kg, s.c.) 7 days prior to the
the median forebrain bundle of rats [11]. More recently, administration of rotenone (10 mg / kg, s.c.) and were
histological, but not biochemical, evidence of selective killed after an additional 7 days. At the end of each
tissue damage has been shown in the striatum and globus experiment, brains were rapidly removed and cut into
pallidus of rats treated for 7–9 days with high doses of 1-mm-thick coronal sections on ice. The striatum was
rotenone as a continuous intravenous infusion [8]. How- carefully excised and placed into 500ml of ice-cold 0.4 N
ever, the possibility that nigrostriatal injury may be perchloric acid. Tissues were disrupted by sonication (10–
induced by rotenone under less severe experimental con- 15 s at 13 mm amplitude in an MSE Soniprep 150
ditions and different paradigms of administration remains sonicator) and centrifuged at 16 0003g for 12 min at 48C.
to be further evaluated. In the present study, changes in Supernatants were removed, stored at2808C and used for
DA levels and metabolism were determined in the mouse DA, 3,4-dihydroxyphenylacetic acid (DOPAC) and
striatum following a single subcutaneous injection of homovanillic acid (HVA) determinations. Pellets were
various doses of rotenone (acute treatment). The effects of resuspended in 500 ml phosphate buffer (pH 7.4) for
the toxicant were also evaluated after multiple systemic protein analysis.
administrations of a relatively mild dose (subchronic
treatment). Finally, mice were exposed to both rotenone 2.3. Assays
and DDC to test the hypothesis that, similar to its effect
with MPTP, DDC may promote or enhance dopaminergic DA, DOPAC and HVA were quantified by reverse-phase
damage by rotenone. HPLC coupled to electrochemical detection (EC)
accord-ing to the method of Kilpatrick et al. [15]. Briefly, samples and external standards were eluted at a flow rate of 1.1
2. Materials and methods ml / min (pump model LC-600 from Shimadzu, Kyoto,
Japan) using a C18 column (Brownlee, 25034.6 mm). The
2.1. Chemicals mobile phase consisted of 90 mM sodium acetate / 35 mM
citric acid (pH 4.7), 130 mM EDTA, 230 mM
1-oc-Rotenone and sodium diethyldithiocarbamate were pur- tanesulfonic acid and 14% methanol. The coulometric
chased from Sigma (St. Louis, MO). MPTP (hydrochloride detector (ESA model 5200A, Chelmsford, MA) consisted
salt) was obtained from Research Biochemicals Interna- of an analytical cell (model 5011) and a guard cell (model
tional BI (Natick, MA). All other reagents were of the 5020). The guard cell and the first and second electrodes of
highest purity available commercially. the analytical cell were set at a potential of 1435, 0 and
1430 mV, respectively. The chromatograms were recorded
2.2. Animals and treatments and integrated (Shimadzu model CR 601) and values are
reported either as percent of their respective controls or as
Seven- to 8-week-old male C57BL / 6 mice (20–25 g, ng / mg of protein.
from Simonsen Laboratories, Gilroy, CA) were maintained For lactate measurements, striata were homogenized in
under a 13:11-h light / dark cycle in a temperature-con- 20ml of a solution containing 20 mM potassium phosphate
trolled room with access to food and water ad libitum. All buffer (pH 6.0), 0.1 M Tris–HCl buffer (pH 8.0) and 0.5
addition of ice-cold 8% perchloric acid (1:2 v / v), the
homogenate was vortexed and centrifuged at 15003g for
10 min at 48C. Lactate was determined in a 10-ml aliquot
of the supernatant using a colorimetric kit purchased from Sigma (St. Louis, MO).
Protein concentrations were determined by the method of Lowry et al. [18] using bovine serum albumin as a standard.
2.4. Statistics
Data were analyzed by a one-way analysis of variance (ANOVA) and, if a P value of less than 0.05 was obtained, pair-wise comparisons were performed using the Student– Newman–Keuls test.
3. Results
When mice were killed 24 h after a single subcutaneous injection of either 5, 10 or 15 mg / kg rotenone, striatal dopamine levels were the same in controls as in rotenone-treated animals. However, rotenone caused a dose-depen-dent increase in striatal DOPAC and HVA concentrations (Fig. 1a). At 7 days, DA remained unchanged while the increase in DA metabolites seen at 24 h was reversed. In fact, DOPAC and HVA levels were slightly reduced in mice treated with 5 or 10 mg / kg rotenone as compared to control animals (Fig. 1b). At 14 days, no significant changes in striatal DA and DA metabolites were measured in mice injected with 10 mg / kg rotenone (Table 1).
A metabolic consequence of mitochondrial electron flow blockage would be the incomplete oxidation of glucose
Fig. 1. Striatal DA metabolism at 24 h (a) and 7 days (b) following a
and consequent lactate accumulation. Thus, in order to single subcutaneous injection of rotenone. HPLC analysis of
catechol-correlate systemic rotenone exposure under our experimen- amines and animal treatments are described in Section 2. Mean6S.E.
a b
values are shown (n54–15). P,0.05 compared to control; P,0.01
tal conditions with tissue alterations in energy metabolism,
c d
compared to control; P,0.001 compared to control; P,0.001
com-striatal lactate levels were measured at 1 and 7 days after a
pared to rotenone at a dose of 5 and 10 mg / kg.
single subcutaneous injection of 10 mg / kg rotenone. A twofold increase in lactate was found at 1 day in
rotenone-The hypothesis that rotenone toxicity may be promoted in as compared to vehicle-treated mice. This effect was
animals with a mild nigrostriatal lesion was tested by completely reversed at the 7 day time point (Table 2).
pre-treating mice with MPTP (15 mg / kg, s.c). The dose of Rotenone was also administered to mice using a
subch-MPTP used for this experiment caused a 40–50% decrease ronic regimen consisting of three weekly injections of 1.5
in striatal DA, DOPAC and HVA. However, subsequent mg / kg for 3 consecutive weeks. A slight decrease in
exposure of these mice to rotenone did not significantly striatal DA and DA metabolites seemed to be caused by
exacerbate MPTP-induced striatal changes (Table 3). rotenone exposure. However, only the decrease in HVA
reached statistical significance (Fig. 2).
The possibility that chemical interactions may affect Table 1
Striatal DA metabolism at 14 days in rotenone (10 mg / kg, s.c.)-treated
rotenone neurotoxicity was investigated next. First,
a mice
rotenone–DDC interactions were studied using the same
paradigm of DDC administration that has been shown to % Control
enhance nigrostriatal damage by MPTP [6,13,31]. DDC DA 10266
was injected intraperitoneally at the dose of 400 mg / kg 2 h DOPAC 9864
HVA 9466
prior to rotenone administration (10 mg / kg, s.c). As shown
a
in Table 3, levels of striatal DA and DA metabolites were Catecholamines analysis and animal treatments are described in Section
Table 2 caused an increase in striatal levels of DOPAC and HVA Striatal lactate content at 24 h and 7 days in rotenone (10 mg / kg, that, paralleling the time course of lactate measurements,
a s.c.)-treated mice
was observed at 1 day and reversed by 1 week. Since the
mmolL-(1)-lactate / mg protein ratio (DOPAC1HVA) / DA is considered a marker of DA Control (n58) 0.0560.002 turnover (with a higher ratio indicating higher turnover),
Rotenone (n55) 24 h 0.1060.02* findings of this study support the conclusion that the 7 days 0.0660.005 primary consequence of acute systemic rotenone exposure a
Lactate analysis and animal treatments are described in Section 2. is an enhancement of DA metabolism and degradation. Mean6S.E. values are shown. *P,0.001 as compared to control. Interestingly, findings obtained with rotenone are similar
to the results reported in previous studies, in which other inhibitors of the mitochondrial respiratory chain were administered to rodents. For example, both sodium azide, a mitochondrial complex IV inhibitor, and 3-nitropropionic acid, a complex II blocker, did not significantly affect striatal DA, while they increased DOPAC and HVA levels when administered systemically to rats [2,4]. The extent of changes in DA metabolites with sodium azide and 3-nitropropionic acid was similar to that measured after rotenone exposure in the present investigation. Thus, it appears that a common feature of intoxication with mito-chondrial poisons is an enhancement of striatal DA turn-over. The mechanisms involved in this effect are currently not well established. However, under conditions of
chemi-Fig. 2. Striatal DA metabolism at 3 weeks following the subchronic cal hypoxia, neuronal depolarization and inhibition of administration of rotenone (1.5 mg / kg s.c., total number of doses59).
energy-dependent re-uptake may lead to DA release and
HPLC analysis of catecholamines and animal treatments are described in
a ultimately to an increase in neurotransmitter turnover
Section 2. Mean6S.E. values of seven animals are shown. P,0.05
[1,7,9,26].
compared to control.
The lack of evidence for dopaminergic terminal injury
4. Discussion after acute rotenone treatment is apparently at odds with a previous report in which striatal DA was depleted
follow-In the present study, the effects of energy metabolism ing stereotaxic injection of rotenone into the rat median
impairment on striatal dopaminergic terminals were as- forebrain bundle [11]. Quite likely, however, differences in
sessed after exposure of mice to rotenone, a known neurotoxicity may reflect differences in the degree of
inhibitor of mitochondrial complex I. Administration of energy impairment caused by rotenone under various
relatively high doses of rotenone using an acute systemic experimental conditions. In particular, direct injection of
model caused metabolic changes consistent with a shift the toxicant into the brain may be expected to cause
from aerobic to anaerobic glucose utilization, as indicated greater energy failure and more pronounced local toxic
by increased striatal lactate levels. These changes were effects. Other experimental variables may underlie
differ-present immediately after rotenone exposure at 1 day, but ences in rotenone-induced dopaminergic injury, including
were reversed within the following week. Rotenone-in- the duration of toxicant exposure. In the present study,
duced energy impairment did not seem to be accompanied mice were also subjected to a mild subchronic regimen
by overt damage to the dopaminergic terminals since DA that, similar to the acute rotenone treatment, did not
concentration was not significantly decreased at any of the produce overt neurotoxicity. In contrast, Greenamyre and
time points examined (1 day to 3 weeks). Rather, rotenone colleagues have recently reported preliminary results
Table 3
a Striatal DA metabolism at 7 days when rotenone or vehicle was administered to mice at 2 h or 7 days after DDC or MPTP, respectively
Treatment DA (ng / mg protein) DOPAC (ng / mg protein) HVA (ng / mg protein)
Control (n515) 143.862.8 12.160.3 14.060.3
´
showing that continuous intravenous infusion of rotenone fellowship from the Fonds de la Recherche en Sante du
´
Quebec (C.T.). caused highly selective dopaminergic lesions that began in
the striatum and progressed retrogradely to the SN [10]. Ferrante et al. have also found histological evidence of
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