Neurochem Res (2010) 35:1402–1412
DOI 10.1007/s11064-010-0198-z
ORIGINAL PAPER
Attenuation of Rotenone-Induced Mitochondrial Oxidative
Damage and Neurotoxicty in Drosophila melanogaster
Supplemented with Creatine
Ravikumar Hosamani • Saraf R. Ramesh •
Muralidhara
Accepted: 15 May 2010 / Published online: 1 June 2010
Ó Springer Science+Business Media, LLC 2010
Abstract Creatine (Cr), an ergogenic nutritional supple-
ment is demonstrated to possess bioenergetic, anti-
excitotoxic and antioxidant properties. This study investigated
the neuroprotective effects of Cr against rotenone induced
oxidative stress, mortality and neurotoxicty in Drosophila
melanogaster. We found significant diminution in the
endogenous levels of oxidative markers in whole body
homogenates of flies exposed to Cr (2–10 mM). Cr sup-
plementation resulted in reduced mortality in flies
exposed to rotenone (500 lM) and better performance in
a negative geotaxis assay. Further Cr (10 mM) markedly
offset rotenone induced mitochondrial oxidative stress,
completely restored the GSH levels, nitric oxide levels,
activity of Mn-SOD and dopamine depletion. In an
oxidative stress bioassay, flies given Cr prophylaxis
exhibited marked resistance to paraquat exposure. These
data allow us to hypothesize that the neuroprotective
action of Cr in Drosophila may be related to its direct
Presented in part during the international conference: 12th European
Drosophila Neurobiology Conference, NEUROFLY 2008’’
September 6th–10th 2008 Wurzburg, Germany (abstract published in
Journal of Neurogenetics special issue, 2009; vol, 23(1): S1–S102.
R. Hosamani  Muralidhara (&)
Department of Biochemistry and Nutrition, Central Food
Technological Research Institute, Council of Scientific and
Industrial Research, Mysore 570020, India
e-mail: mura16@yahoo.com
S. R. Ramesh
Department of Studies in Zoology, Manasagangotri, University
of Mysore, Mysore 570020, India
123
antioxidant activity and ability to abrogate rotenone
induced mitochondrial oxidative stress.
Keywords Creatine  Drosophila  Oxidative stress 
Mitochondria  Dopamine  Neurotoxicty
Introduction
Creatine (Cr), a natural substance in the human body, is
partly synthesized endogenously as well as ingested
exogenously from food especially meat and fish. It plays a
vital role in providing rapid energy during muscle con-
traction which involves the transfer of N-phosphoryl group
from phosphorylcreatine (PCr) to ADP to regenerate ATP
through a reversible reaction catalyzed by phosphorylcre-
atine kinase (PCK) [1]. Cr chiefly functions to transfer
energy from mitochondria to cytosol. The PCr-PCK system
serves as an energy buffer by connecting the mitochondrial
sites of energy production with cytosolic sites of energy
consumption in tissues with high energy demand such as
brain and muscle [2]. Of the various physiological func-
tions assigned to the PCr-PCk system in vivo, prevention of
oxidative stress via direct and indirect antioxidant action is
one of the major roles [3].
Currently Cr is widely used as an ergogenic nutri-
tional supplement regularly by athletes to improve
muscular performance [4]. Several studies have con-
firmed the beneficial effects of Cr supplementation in
patients suffering from atrophy, muscle weakness, and
metabolic dysfunctions. Recent investigations have
revealed the potential therapeutic effects of Cr supple-
mentation such as—improved brain function among
young subjects and elderly people [5], attenuation of
stress-induced cognitive impairment and reduction of
Neurochem Res (2010) 35:1402–1412
mental fatigue [6]. Several evidences indicate specific
protective effects of Cr in various animal models of
neurodegenerative diseases or ischemic stroke [7–9]. The
neuroprotective effects of Cr has been ascribed to the
buffering capacity of cellular ATP levels coupled with
mitochondrial targeted antioxidant properties in cell and
mammalian models [10–12].
Non-mammalian model such as Drosophila melano-
gaster has been extensively exploited as a powerful
genetic tool to understand complex biological problems.
In recent times, numerous researchers have utilized this
model to understand various neurodegenerative diseases
[13, 14]. Proteomic analyses have revealed that greater
than 70% of the disease-related loci in humans have a
clear orthologs in Drosophila [13]. The high degree of
conservation revealed through proteomic analyses, the
presence of a complex nervous system in an intact
organism amenable to genetic manipulation, and the rel-
atively short lifespan of the flies make Drosophila ideal
for studying progressive neurological conditions including
Parkinson’s disease (PD). Familial forms of PD have led
to the discovery of affected genes and important mecha-
nistic insight into the disease. However, the majorities of
cases are sporadic of unknown etiology or thought to be
the result of exposure to environmental toxins such as
rotenone, paraquat etc. [15, 16].
Rotenone, a high affinity specific inhibitor of mito-
chondrial complex I is capable of causing mitochondrial
dysfunctions that phenocopies PD [17–19]. Chronic rote-
none treatment has been used in Drosophila to create a
pharmacological model of PD and induce both dopami-
nergic cell loss and locomotor impairments [13, 17].
Although Drosophila is employed to elucidate mechanistic
basis of various neurological disorders, it is less employed
for evaluating potential therapeutic interventions. Since
Drosophila allows rapid screening of putative neurothera-
peutic agents, we have employed this model to obtain
mechanistic insights of specific neuroprotective com-
pounds under oxidative stress mediated neurotoxicty [20].
In this study, we examined the neuroprotective action of
creatine against rotenone induced mortality, mitochondrial
oxidative stress and neurotoxicty in Drosophila. We found
that Cr supplementation in flies not only reduced the
incidence of rotenone induced mortality, but also signifi-
cantly abrogated mitochondrial oxidative stress, restored
the depleted dopamine levels and attenuated rotenone-
induced neurotoxicity as determined in a negative geotaxis
assay. Further, flies given Cr prophylaxis exhibited resis-
tance against paraquat intoxication. Based on our results
we hypothesize that the neuroprotective action of creatine
in Drosophila may be largely ascribed to the combined
action as an antioxidant and its ability to mitigate mito-
chondrial oxidative stress.
1403
Experimental Procedure
Materials
Creatine monohydrate, Rotenone, Paraquat, Dopamine,
Thiobarbituric acid (TBA), 1,1,3,3-tetramethoxypropane,
2,7, dichloro-fluoroscein (DCF), 2,7, dichlorofluorescein
diacetate (DCF-DA), and Griess reagent were procured
from Sigma Chemical Co. St Louis, USA. All other
chemicals used were of analytical grade.
Drosophila Culture and Husbandry
Synchronized Drosophila melanogaster, wild (Oregon K),
adult male flies (8–10d old) were obtained from the
Drosophila stock culture facility at Manasagangothri,
University of Mysore, Karnataka. The flies were main-
tained at 25 ± 1°C and 70–80% relative humidity and fed
on a standard wheat flour-agar diet with yeast granules.
Diet was prepared according to a standard protocol [20].
Male adult flies were isolated by following a standardized
protocol. In brief, a given number of flies are exposed to
few drops of diethyl ether in a small airtight glass container
for 1 min. In case of over anesthetization, flies will be
found dead with their wings stretched perpendicular to the
body axis.
Preliminary Study
Preliminary studies were conducted with small fly numbers
with three concentrations of Cr viz., 2, 5 and 10 mM to
determine the effect of Cr alone on the survival of the flies
during the experimental period. However, for the deter-
minative studies such as mortality and climbing assay, only
two concentrations viz., 5 and 10 mM per unit of medium
were chosen. Further, for co-exposure study only one
concentration of Cr viz., 10 mM was employed. The
selection of 10 mM concentration was based on recent
findings in cell models [41] and our own observation of
maximum reduction of endogenous MDA levels in flies.
Rotenone (Rot) Exposure
In a preliminary study, flies were exposed to rotenone at
concentrations of 250, 500 and 1000 lM for 7 days to
determine lethality. However, only one concentration of
rotenone (500 lM) was employed to assess the neuropro-
tective effect of Cr. For these studies, rotenone exposed
flies were provided with Cr (10 mM) in the diet and the
modulatory effect of Cr on rotenone-induced lethality,
locomotor dysfunctions, dopamine levels, oxidative
impairments in both cytosol and mitochondria were
determined. For mortality studies, a minimum of 50 adult
123
1404
flies per replicate (three replicates) were exposed to Cr or
rotenone or combination of Cr and rotenone for 7 days.
Lethality profile of the flies were recorded every 24 h till
the end of the experiment and expressed in terms of percent
mortality. Similarly, behavioral and biochemical investi-
gations were also carried out.
Biochemical Measurements
Following various treatments, flies were mildly anesthe-
tized using diethyl ether in a small airtight glass container
for 1 min. Quantification of oxidative markers viz., Mal-
ondialdehyde (MDA), hydroperoxide (HP) levels, reduced
glutathione (GSH) content, total thiols (TSH) and activities
of antioxidant enzymes viz., catalase and superoxide dis-
mutase (SOD) were determined only in whole body
homogenates. However, the dopamine levels were deter-
mined in head and body region of flies. Further, mito-
chondrial oxidative impairments were assessed in terms of
reactive oxygen species (ROS) generation, nitric oxide
(NO) levels and activities of complexes I–III and complex
II–III were determined. In addition, cholinergic functions
were measured in terms of the activities of acetylcholin-
esterase (AChE) and butyrylcholinesterase (BChE) in
whole body homogenates.
Isolation of Cytosol and Mitochondria from Flies
Whole body homogenate/cytosol was prepared by using
sodium-phosphate buffer, (0.1 M, pH 7.4). Following
homogenization, samples were centrifuged at 25009g for
10 min at 4°C, supernatant filtered through nylon mesh
(pore size, 10 lm) and used as cytosol for further bio-
chemical assays. Mitochondria from whole body of flies
were prepared by differential centrifugation. Briefly, fly
homogenate (4%) was prepared in ice-cold Tris–sucrose
buffer (Tris buffer, 2 mM and Sucrose, 250 mM) of
0.25 M, pH 7.4 using a glass-Teflon grinder, and filtered
through 10 lM tissue sieve. The filtrate was centrifuged
(25009g for 10 min), pellet was discarded and supernatant
further subjected to centrifugation at 78009g for 10 min to
obtain the nuclear pellet. Mitochondria were obtained by
centrifuging the post-nuclear supernatant at 10,0009g for
10 min. The pellet was washed mannitol-sucrose-HEPES
buffer (Mannitol, 200 mM; Sucrose, 70 mM; EDTA,
0.1 mM and HEPES, 10 mM) and resuspended in the
buffer, stored at -20°C until future use.
Preparation of Homogenate from Head and Body
Regions for Dopamine Estimation
Known numbers of fly heads were separated from rest of
the body using a sharp blade. 20 heads and body regions
123
Neurochem Res (2010) 35:1402–1412
were homogenized in sodium-phosphate buffer (0.1 M, pH
7.4), followed by centrifugation at 25009g for 10 min at
4°C. Supernatant was used directly to estimate the dopa-
mine level by HPLC method [21].
Assay for Lipid Peroxidation and Hydroperoxide
Levels
Lipid peroxidation was measured by employing thiobar-
baturic acid (TBA). Briefly, the reaction mixture contained
500 ll fly homogenate, 1.5 ml acetic acid (pH 3.5, 20%),
1.5 ml of TBA (0.8% w/v), 200 ll sodium lauryl sulphate
(SDS) (8% w/v). The mixture was heated in a boiling water
bath for 45 min and adducts formed were extracted into
3 ml of 1-butanol. The absorbance was measured at
532 nm and quantified as malondialdehyde equivalents
using 1,1,3,3-tetramethoxypropane as the standard [22].
Hydroperoxide generation was determined according to the
method of Wolff [23]. An aliquot of homogenate (100 lg
protein) was added to 1 ml of FOX reagent (250 lM Fer-
rous ammonium sulphate; 100 mM sorbitol; 25 mM
H2SO4; 100 lM xylenol orange), and incubated for 30 min
at room temperature. The absorbance was taken at 560 nm
and expressed as nmoles hydroperoxides/mg protein.
Determination of Reduced Glutathione and Total Thiols
Reduced glutathione (GSH) content was estimated
according to the fluorimetric method employing o-phthal-
aldehyde (OPT). An aliquot of whole body homogenate
was added to 0.1 M formic acid and spun at 52009g for
10 min to precipitate protein, then the supernatant was
allowed to react with OPT (1 mg/ml in methanol) at room
temperature for 30 min and fluorescence measured at
excitation of 345 nm and emission at 425 nm [24]. To
estimate total thiols, 200 ll of homogenate (400 lg pro-
tein) was added to 300 ll of tris–buffer (2 mM, pH 8.2),
25ul of 5, 50 -dithiobis 2-nitrobenzoic acid (DTNB) (10 mM
in methanol) and 1.975 ll of methanol and allowed to
stand for 30 min for room temperature with occasional
shaking. Following centrifugation at 30009g for 15 min,
the supernatant was read at 412 nm against distilled water
blank and calculated using molar extinction coefficient
(MEC) 13.6 mM-1 cm-1 [25].
Enzyme Activities: Catalase and Superoxide Dismutase
Catalase activity was measured following the method of
Aebi [26]. To 1 ml reaction mixture containing 8.8 mM
H2O2 (3%), 0.1 M sodium phosphate buffer, pH 7.0. The
reaction was initiated by adding an aliquot (equivalent to
10 lg protein). The decrease in H2O2 was monitored for
3 min at 240 nm and expressed as lmol of H2O2
Neurochem Res (2010) 35:1402–1412
decomposed/min/mg protein (MEC-H2O2 44.1 mM-1
cm-1). SOD activity was determined by monitoring inhi-
bition of quercetin auto oxidation. Total volume of 1 ml
reaction mixture containing 3–5 lg protein, 0.016 M
sodium phosphate buffer, pH 7.8; N,N,N,N tetramethyl
ethylenediamine (TEMED), 8 mM and ethylenediamine-
teraacetic acid (EDTA, 0.08 mM) and reaction was started
by adding 0.15% quercetin dissolved in dimethyl form-
amide (DMF). Reaction was monitored for 3 min at
406 nm, expressed as amount of protein required to inhibit
50% of quercetin auto oxidation [27].
Activities of Acetylcholinesterase (AChE)
and Butyrylcholinesterase (BChE)
AChE and BChE activities were determined according to
the method of Ellmann [28]. To the reaction mixture
containing 1 ml phosphate buffer (0.1 M, pH 8.0), 5,5-
dithiobis 2-nitrobenzoic acid (DTNB,10 mM), sample
(cytosol) and acetylthiocholineiodide (78 mM)/butyrylthi-
ocholine iodide were added and the change in absorbance
was monitored at 412 nm for 3 min. The enzyme activities
were expressed as nmoles of substrate hydrolyzed/min/mg
protein.
Assays in Mitochondria: Activities of NADH-
Cytochrome C Reductase (Complex I–III)
and Succinate-Cytochrome C Reductase (Complex II–III)
Mitochondria isolated from whole body of adult flies
(50 lg) were added to phosphate buffer (0.1 M, pH 7.4)
containing NADH (0.2 mM) and KCN (1 mM). The reac-
tion was initiated by addition of 0.1 mM cytochrome C and
decrease in absorbance was monitored for 3 min at 550 nm.
The activity was expressed as lmol cytochrome C reduced/
min/mg protein (molar extinction coefficient (MEC)
-19.6 mM/cm) [29]. An aliquot of mitochondria from
whole body (50 lg) were added to phosphate buffer (0.1 M,
PH 7.4, 2 mM EDTA) containing succinate (20 mM) and
KCN (1 mM). The reaction was initiated by addition of
0.1 mM cytochrome C and decrease in absorbance was
monitored for 3 min at 550 nm. The activity was expressed
as lmol cytochrome C reduced/min/mg protein (molar
extinction coefficient (MEC) -19.6 mM/cm) [29].
Reactive Oxygen Species (ROS) and Nitric Oxide (NO)
Levels
ROS was assayed using dihydro dichlorofluorescein diac-
etate. Briefly, the reaction mixture (2 ml) containing
Locke’s buffer (Nacl, 154 mM; Kcl, 5.6 mM; NaHCo3,
3.6 mM; HEPES, 5 mM; CaCl2, 20 mM; D-Glucose,
1405
10 mM) pH 7.4, 0.1 ml homogenate (100 lg protein) and
10 ll of DCFH-DA (5 lM) was incubated for 15 min at
room temperature to allow the DCFH-DA to be incorpo-
rated into any membrane bound vesicles and diacetate
group to be cleaved by esterases. After 15 min of further
incubation, the conversion of DCFH-DA to the fluorescent
product DCF was measured in a spectrofluorometer with
excitation wavelength of 484 nm and emission at 530 nm.
Background fluorescence was corrected by the inclusion of
parallel blanks. ROS formation was quantified from a DCF
standard curve and data are expressed as pmoles DCF
formed/min/mg protein [30].
Nitric oxide levels were measured by using commer-
cially available Griess reagent (1.5% Sulphanilamide and
0.15% N-1-naphthyl-ethylene diamine in 1 N HCl) pro-
cured from M/s Sigma Chemicals (St. Louis, MO, USA).
The principle of assay is based on the enzymatic conver-
sion of nitrate to nitrite by nitrate reductase. The reaction is
based on two step diazotization reaction in which acidified
NO2 produces a nitrosating agent which reacts with sul-
phanilic acid to produce the diazonium ion which is cou-
pled with NED, N-(1-napthylethylenediamine to form the
chromophoric azo derivative which absorbs light at
540 nm. Nitric oxide levels were quantified from a sodium
nitrate standard curve [31].
Determination of Dopamine Levels by HPLC
Head/rest of the body were homogenized in 100/500 ll of
ice cold 0.1 M phosphate buffer (pH, 7.4) containing
1 mM EDTA. Following centrifugation at 25009g for
10 min, the supernatant was filtered through sieve and
injected directly into HPLC column (C-18 column reverse
phase, Linchrospher 100RD-18, 14.5 cm, 5 lm, E. Merck)
equipped with ultraviolet detector set at 280 nm. Mobile
phase consisted of 0.2% aqueous trifluoroacetic acid and
methanol (70:30 v/v) and the flow rate was maintained at
1 ml/min [20, 21].
Negative Geotaxis Assay (Climbing Assay)
Test flies were anesthetized and placed in a vertical glass
column (standard length, 25 cm; diameter, 1.5 cm). After a
brief recovery period, flies were gently tapped to the bot-
tom of the column. Following 1 min, flies that reached the
top of the column and flies that remained at the bottom
were counted separately. Data was expressed as percent
flies escaped beyond minimum distance of 6 cm in 60 s of
interval [32]. Twenty adults per replication were used for
assay. The assays were repeated three times and the score
for each replication was an average of three such trials for
each group of insects including control.
123
1406
Paraquat Resistance Test
Paraquat is a well known in vivo free radical generator.
The ‘paraquat resistance’ test has been employed to mea-
sure acquired resistance to oxidative stress. Flies were
exposed to paraquat for 48 h (20 and 40 mM, in 5%
sucrose solution) as described previously [20, 33]. The rate
of survival of flies among untreated controls and Cr treated
group was recorded during 72 h of observation period.
Determination of Protein
Protein concentrations in the cytosol and mitochondria of
whole body flies were determined by Lowry’s method [34]
using bovine serum albumin as the standard.
Statistical Analysis
Results are represented as the group means ± standard
error (SE) for each experimental group. The data was
analyzed using one way ANOVA followed by post hoc
‘Tukey’ test and P value less than 0.05 was set as the
minimum level of significance.
Results
Effect of Creatine (Cr) on Endogenous Markers
of Oxidative Stress and Cholinergic Function
A concentration related diminution in both MDA and
hydroperoxide levels was observed in whole body
homogenates of adult flies fed with Cr supplemented diet
for 7 days (Table 1). The reduction in MDA levels were
more marked (17–44%) compared to the reduction in HP
levels (17–21%). However, Cr did not have any effect on
GSH and total thiols content in whole body homogenates.
Likewise, the activities of antioxidant enzymes viz., Cat-
alase and Superoxide dismutase also showed no significant
alterations. The activities of both acetylcholinesterase
and butyrylcholinesterase enzymes among flies fed with Cr
for 7 days remained unchanged at all Cr concentrations
tested (Table 1).
Protective Effect of Creatine Against Rotenone-
Induced Neurotoxicty
Modulatory Effect of Creatine against Rotenone induced
lethality
Exposure of adult flies to Rotenone resulted in a concen-
tration dependent lethality over a 7-day experimental per-
iod (Fig. 1a). Neurotoxicant induced deaths occurred
123
Neurochem Res (2010) 35:1402–1412
between 4–7 days and terminally the cumulative percent
lethality was: 250 lM-14%; 500 lM-55%; 1000 lM-78%.
In order to examine the modulatory effect of creatine, we
used only one concentration of rotenone (500 lM) and two
concentrations of creatine. Interestingly, co-exposure of
flies with creatine (5 and 10 mM) resulted in lower inci-
dence of mortality among rotenone exposed flies. The
degree of protection was robust (74 and 52%) (Fig. 1b)
indicating its ability to protect against rotenone induced
lethality.
Creatine Protects Rotenone-Induced Locomotor Deficits
Data obtained in the negative geotaxis assay in flies
exposed previously to rotenone for 7 days revealed con-
centration dependent locomotor dysfunctions (Fig. 2a). At
higher concentrations of rotenone, more number of flies
had a tendency to stay at the bottom of vial. Among
untreated controls, more than 94% flies were able to reach
at the top of the vial within a minute, while rotenone
exposed flies exhibited significant decrease in climbing
ability with increasing concentration of rotenone (250 lM,
44%; 500 lM, 19% and 1000 lM, 15%) clearly suggesting
the induction of locomotor deficits. In a parallel experi-
ment, we also measured the modulatory effect of Cr on
rotenone induced locomotor deficits in a similar paradigm.
Rotenone treated flies exhibited severe locomotor impair-
ments, while co-exposure with creatine significantly
improved (35 and 45%) the performances of flies (Fig. 2b)
in the negative geotaxis test. In general, co-exposed flies
appeared to be more active than rotenone alone treated
flies. Further, Cr at both the tested concentration alone had
no significant effect on locomotor behavior among flies.
Effect on Rotenone-Induced Depletion of Dopamine (DA)
Levels
Cr alone treatment did not significantly affect the DA
levels measured either in head or body homogenates of
flies (Fig. 3). In contrast, rotenone caused robust DA
depletion in both the regions (head, 41%; body region,
70%). Interestingly, the degree of rotenone induced DA
depletion was less robust among flies co-exposed to Cr.
Restoration of DA levels affected by Cr treatment was 50%
in head and 28% in rest of the body region of flies.
Effect of Creatine Against Rotenone-Induced
Mitochondrial Oxidative Stress
Effect on ROS Generation and GSH Levels
Flies fed with Cr alone for 7d showed marginal (14%)
decrease in endogenous ROS levels. On exposure to
Neurochem Res (2010) 35:1402–1412 1407

Table 1 Effect of Creatine (Cr) supplementation for 7 days on the endogenous markers of oxidative stress, activities of antioxidant enzymes and
cholinergic function in whole body homogenates of adult Drosophila melanogaster
Parameters Creatine (mM)
0 2 5 10

Malondialdehydea 3.51 ± 0.32 2.91 ± 0.23 2.26 ± 0.11* 1.98 ± 0.36*

Hydroperoxideb 0.52 ± 0.08 0.42 ± 0.009 0.43 ± 0.019* 0.41 ± 0.062*
Glutathionec 31.33 ± 0.89 34.34 ± 2.14 36.09 ± 1.02* 32.30 ± 2.27
Total thiolsd 0.07 ± 0.005 0.064 ± 0.004 0.076 ± 0.003 0.067 ± 0.006
SODe 204.2 ± 1.73 244.1 ± 18.2 253.2 ± 38 238.2 ± 28.3
f
Catalase 0.174 ± 0.01 0.178 ± 0.003 0.171 ± 0.014 0.136 ± 0.005
Acetylcholinesteraseg 0.080 ± 0.001 0.084 ± 0.005 0.083 ± 0.0034 0.076 ± 0.009
Butyrylcholinesteraseg 0.041 ± 0.002 0.041 ± 0.002 0.043 ± 0.002 0.038 ± 0.004
Adult flies were provided with creatine in feed at concentrations of 2, 5 and 10 mM for 7 days. All biochemical parameters were quantified in
whole body homogenates
Values are mean ± SE (Three sets of flies). Data analyzed by one way ANOVA followed by post hoc ‘Tukey’ test (*P \ 0.05)
a
gmol MDA/mg protein; bgmol hydroperoxides/mg protein; clg GSH/mg protein; dmmol DTNB/min/mg protein; eUnits/min/mg protein; flmol
H2O2 hydr/min/mg protein; gqmol DTNB/min/mg protein
For more details, see ‘‘Material and Methods’’

A 100 rotenone, mitochondria obtained from whole body

Rotenone 250µM Rotenone 500µM
homogenate of flies revealed marked (43%) increase in
Rotenone 1000µM
80 ROS levels. Interestingly, co-exposure with Cr completely
Mortality (%)

abrogated the rotenone induced ROS generation as

60
revealed by near normal levels of ROS (nearly 90% pro-
tection) (Fig. 4a). While rotenone only treated flies showed
40
significant (31%) depletion of GSH levels, flies co exposed
to creatine exhibited normal GSH levels (Fig. 4b). Further,
20
GSH levels among Cr alone treated flies remained
0 unaltered.
96 120 144 168
Exposure time (hrs) Effect on the Activity of Mn-SOD and Nitric Oxide (NO)
Levels
B 100

80 Cr treatment alone did not alter the activity levels of

Mortality (%)

Mn-SOD enzyme measured in mitochondria. However,

60
rotenone alone treated flies showed elevated (by 19%) SOD
40 activity. Surprisingly, co-exposed (rotenone and creatine)
20
flies showed normalized activity compared to rotenone
treated group (Fig. 5a). Flies exposed to Cr (10 mM) alone
0 showed significantly decreased endogenous nitric oxide
M

ot

ot
M
l
tro

0m

levels (by 33%). However, rotenone exposure resulted in

0µ
m

R
on

r5

50

+
r1

M
C

significant increase in nitric oxide levels (29%). Interest-

C

ot

0m
r5
R

r1
C

ingly, Cr supplementation resulted in complete restoration

C

of NO to normal levels (Fig. 5b).

Fig. 1 Rotenone (Rot) induced mortality response in Drosophila
melanogaster and its modulation by creatine (Cr) supplementation.
a Mortality pattern among adult Drosophila exposed to different Effect on the Activities of ETC Enzymes
concentrations of Rotenone in feed (250, 500 and 1000 lM).
Rotenone exposure caused a concentration dependent lethality. Cr alone fed flies showed no change in the complex I–III
b Modulation of Rotenone induced mortality among flies co-exposed
with creatine. Cr supplementation significantly reduced the incidence
activity compared to control. However, significant inhibi-
of Rotenone induced mortality tion (29%) was evident among rotenone only treated flies.

123
1408 Neurochem Res (2010) 35:1402–1412

A 100 Head Rest of body

µg dopamine/mg protein
15
* *
80
% Flies escaped/min

12

9
60
6
* *
40 3

M
20

ot

ot
ol

ol
M

M
0µ

0µ
0m

0m
tr

tr
+R

+R
on

on
50

50
M

M
r1

r1
C

C
0m

0m
ot

ot
C

C
R

R
r1

r1
0

C
0 250 500 1000
Fig. 3 Modulatory effect of creatine (Cr) supplementation on
Rotenone (µM) Rotenone (Rot)-induced dopamine depletion among Drosophila
melanogaster determined in homogenates prepared from head and
B body region separately. Cr supplementation significantly restored
100
Rotenone induced dopamine depletion in both head and body regions.
% Flies escaped/min

Values are mean ± SE (in triplicates), data was analyzed by one way
80
ANOVA followed by post hoc Tukey test (*P \ 0.05). Significances
were determined by making comparisons between control v/s Cr, Rot;
60
Rot v/s Rot ? Cr. (n = 50 flies per replicate, three such replication
used for assay)
40

20 supplemented flies were able to survive for longer time

compared to rotenone alone treatment at both observation
0
times (Fig. 6). The percent protection at 48 h was—33 and
ol

ot

ot
M
M

52% and at 72 h; 49 and 47% at both 20 and 40 mM of PQ,

tr

0µ

R
0m
on

r5

+
50
r1

M
C

respectively.
M
C

0m
ot

m
C

r5

r1
C

Fig. 2 Rotenone (Rot) induced locomotor deficits (expressed as

percent flies escaped) and its modulation by creatine (Cr) supple-
Discussion
mentation determined in a negative geotaxis assay. a Rotenone
exposure resulted in a concentration dependent locomotor impairment Although there is lack of certainty on the pathophysiology
in adult Drosophila melanogaster. b Modulation of Rotenone induced of neurodegenerative mechanisms, it is well accepted that
locomotor deficits among flies given Cr supplementation. (n = 50
flies per replicate, three such replication used for assay). Cr
energy depletion, oxidative stress and mitochondrial dys-
supplemtation markedly improved the performance of Rotenone functions are vital factors associated with most of these
treated flies in the negative geotaxis assay disorders [35, 36]. Drosophila is widely used as a model to
understand the pathophysiolgy of several neurodegenera-
Interestingly, the activity levels of complex I–III were tive diseases (NDD). In recent times it is also effectively
completely restored among Cr co-exposed flies (Table 2). serving as an easy platform to screen several phyto-
Further, Cr alone fed flies showed no change in the activity chemicals for their neuroprotective properties and is
level of complex II–III enzyme. Furthermore, rotenone anticipated to be highly valuable for first-step drug screen.
alone and co-treatment with creatine also failed to modu- Previously, using Drosophila we demonstrated the pro-
late the activity level of complex II–III. tective effects of Bacopa monnieri against rotenone
induced neurotoxicty [20]. In this study we tested the
Creatine Prophylaxis Confers Resistance to Paraquat hypothesis that creatine supplementation can significantly
Exposure offset rotenone induced mitochondrial oxidative stress in
Drosophila and rescue the flies from the neurotoxic
Initially, we determined the LC50 concentration of paraquat consequences.
among adult flies. At 24 h in both the tested concentration Creatine has been widely used as an ergogenic aid to
no significant mortality was recorded. However, significant improve exercise performance in humans and also to
mortality was evident at 48 and 72 h. The incidence of ameliorate oxidative stress mediated diseases. However,
mortality was: 15, 42% at 48 h and 39 & 95% at 72 h the underlying mechanism/s of its action are less well
at both the concentration, respectively. Interestingly, Cr understood in vivo [37–39]. In the present study, flies

123
Neurochem Res (2010) 35:1402–1412
* *
A
0.20
picomole dichloro flouroscein
formed/mg protein/min
0.15
0.10
0.05
0.00
Control Cr 10mM Rot 500µM Cr +Rot
B 100 *
*
µg GSH/mg protein
75
50
25
0 Control
Control Cr 10mM Rot 500µM
Fig. 4 Modulatory effect of creatine (Cr) supplementation on
Rotenone (Rot)-induced oxidative stress measured as reactive oxygen
species (ROS) (a) and reduced glutathione (GSH) (b) levels in whole
body mitochondrial fraction of Drosophila melanogaster. Cr supple-
mentation significantly protected against Rotenone induced ROS
generation and GSH depletion. Values are mean ± SE. Data was
analyzed by one way ANOVA followed by post hoc Tukey test
(*P \ 0.05). Significance was determined by making comparisons
between control v/s Cr, Rot; Rot v/s Rot ? Cr. (n = 50 flies per
replicate, three such replication used for assay). For more details, see
‘‘Material and Methods’’
provided with Cr supplements exhibited significant reduc-
tion in the endogenous levels of oxidative markers such as
malondialdehyde and hydroperoxides. While the lowest
concentration of Cr had no appreciable effect, at higher
concentrations significant diminitution of MDA and
hydroperoxide levels in whole body homogenates sug-
gested its potential to modulate endogenous levels of oxi-
dative markers. These findings of diminished levels of
oxidative markers associated with enhanced GSH levels in
whole body homogenates clearly suggest the antioxidative
effects of Cr. Owing to higher reduction in MDA levels
observed with 10 mM Cr, we employed this concentration
for our further experimentation. The observed effects in
flies are consistent with previous reports on the direct
antioxidant properties of Cr both under in vitro and in vivo
conditions. Previously the potential of Cr to scavenge
1409
A 600
* *
Units/min/mg protein
Mn-SOD activity
400
200
0
Control Cr 10mM Rot 500µM Cr +Rot
B 0.8
* *
Absorbance/mg protein
0.6
Nitrite formed
0.4
0.2
0
Cr 10mM Rot 500µM Cr +Rot
Cr +Rot
Fig. 5 Modulatory effect of creatine (Cr) supplementation on
mitochondrial Superoxide dismutase activity (Mn-SOD) (a) and
nitric oxide (NO) levels (b) in whole body mitochondrial fractions of
Drosophila melanogaster exposed to Rotenone. Mitochondrial SOD
activity and NO levels were markedly restored to near normalcy
among flies given Cr Supplementation. Values are mean ± SE (in
triplicates), data was analyzed by one way ANOVA followed by post
hoc Tukey test (*P \ 0.05). Significances were determined by
making comparisons between control v/s Cr, Rot; Rot v/s Rot ? Cr.
(n = 50 flies per replicate, three such replication used for assay). For
more details, see ‘‘Material and Methods’’
ABTS?, superoxide anions and peroxynitrite radicals were
demonstrated in vitro [40]. Studies in cell models also
demonstrated the direct antioxidant activity of Cr via a
scavenging mechanism in oxidatively injured mammalian
cells and its DNA protective action against oxidative attack
[41, 42]. Further long term Cr supplementation in aged
mice was reported to diminish the ROS levels, decrease the
accumulation of lipofuscin pigments in brain and extend
longevity [43]. In a recent study, Cr supplementation was
shown to reduce endogenous lipid peroxidation biomarkers
in rats suggesting a protective role against oxidative dam-
age [44].
Rotenone, a respiratory chain complex I inhibitor is
hypothesized to be a vital environmental toxin (along with
other chemicals such as MPTP and Paraquat) contributing
significantly towards the occurrence of sporadic PD in
humans [45]. Chronic exposure of Drosophila to sub lethal
123
1410 Neurochem Res (2010) 35:1402–1412

Table 2 Effect of dietary supplementation of Creatine (Cr) (10 mM) Earlier findings in Drosophila viz., prevention of rotenone
against rotenone (500 lM) induced mitochondrial dysfunctions induced locomotor deficits and DA neuronal loss by the
measured in whole body mitochondrial fractions of adult Drosophila
antioxidant melatonin clearly suggest the involvement of
melanogaster
oxidative stress mechanisms [47]. Interestingly, similar
Groups Enzyme activity signs of oxidative damages have been reported frequently
Complex I–IIIa Complex II–IIIb in dopaminergic neurons from PD patients, suggesting
implication of oxidative stress in this disease [48, 49]. In
Control 0.059 ± 0.008 0.048 ± 0.003
the present study, rotenone induced significant oxidative
Creatine, 10 mM 0.049 ± 0.009 0.040 ± 0.003
markers as evidenced by enhanced MDA and hydroper-
Rotenone, 500 lM 0.042 ± 0.005* 0.043 ± 0.001
oxide levels, elevated activities of antioxidant enzymes
Creatine ? rotenone 0.064 ± 0.004* 0.037 ± 0.001 (Catalase and SOD). These findings are consistent with our
Adult flies were exposed to rotenone (500 lM) with or without cre- own findings in Drosophila [20] and previous reports in
atine (10 mM) for 7 days and mitochondria isolated from whole body rats exposed to rotenone [50, 51]. Severe depletion in
homogenates were assayed for the activities of ETC enzymes; Values
cellular GSH levels upon rotenone exposure in Drosophila
are mean ± SE (Three sets of flies). Data analyzed by one way
ANOVA followed by post hoc ‘Tukey’ test (*P \ 0.05) adds further evidence that a state of oxidative stress exists
a, b
lmol cytochrome c reduced/min/mg protein in vivo which may lead to mitochondrial damage and
For more details, see ‘‘Material and Methods’’ increase in free radical generation.
Cr supplementation restored the depleted GSH pool and
normalized the ROS levels in rotenone treated flies. This
* * protective action may be chiefly attributable to the direct
100 quenching of free radicals or alternatively due to the up
regulation of antioxidative defenses. While the primary
80 48 hrs 72 hrs effect of Cr supplementation on GSH synthesis in flies
needs further investigations, it may be the result of de novo
Mortality (%)

60
synthesis of GSH and phase II detoxification in response to
* *
* * rotenone induced altered redox status. The lower incidence
of mortality among Cr supplemented flies clearly suggests
40
the protective action of Cr. Furthermore, rotenone induced
* neurotoxicty was clearly evidenced by high rate of loco-
20 motor deficits measured in the negative geotaxis assay.
Flies with locomotor deficits have tendency to stay at the
0 bottom of vial and do not appear to coordinate their legs in
normal fashion. The development of this phenotypic
PQ mM

PQ mM
M

M
ol
M

M
ol

PQ mM

PQ M

M
M
m

m
0m

0m
tr
tr

m
m

on
on

40
40
20

20
20

20

40
40
r1

r1

expression of among flies exposed to rotenone is speculated

C

C
PQ

PQ
PQ

PQ
C

C
+

to be due to the deficits in high energy levels of ambulatory

+

M
M

M
M

0m
0m

0m
0m

and flight muscles which are rich in mitochondria. Hence it

r1

r1

r1

r1
C

is likely that uncoupled mitochondrial machinery may be

Fig. 6 Paraquat (PQ) induced mortality response in Drosophila
melanogaster and its modulation by creatine (Cr) prophylaxis. Time
course lethality response expressed as percent mortality among flies
exposed to various concentrations of PQ and its modulation by Cr
prophylaxis (n = 50 flies per replicate, three such replication used for
assay). For more details, see ‘‘Material and Methods’’
doses of rotenone is well demonstrated to recapitulate the
main symptomatic features of PD viz., a selective loss of
dopaminergic neurons including locomotor defects [17,
46]. In the present model, rotenone induced a concentration
related mortality and locomotor deficits in adult flies, data
which is consistent with previous reports [13, 46]. While
the exact mechanism of rotenone action leading to neuro-
toxicity is not well understood, in the insect model, rote-
none action is attributed to specific sensitivity of
dopaminergic neurons to ROS and oxidative damage [45].
responsible of the induced locomotor dysfunctions as evi-
denced by severe complex I inhibition. Another possible
reason might be due to the differential and significant
depletion of dopamine pool as observed in both head and
body homogenate. Interestingly Cr supplementation was
able to rescue the flies significantly from deteriorating
locomotor dysfunctions indicating its potential to restore
dopamine pool and protect the mitochondrial function. This
thinking is consistent with earlier findings which suggest
significant correlation between locomotor dysfunction and
dopamine deficiency [13]. Although speculative, the better
performance resulting from Cr supplements among rote-
none exposed flies could be partly related to its well known
physiological role in the maintenance of ATP/ADP ratio
resulting in enhanced mitochondrial respiration [1, 52].
However further investigations are necessary to delineate

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Neurochem Res (2010) 35:1402–1412
the direct effects of Cr on flight muscles among flies
exposed to rotenone.
Employing a prophylactic approach, we obtained further
evidence that Cr has the potential to confer protection
against paraquat induced oxidative insult. Paraquat, a well
known herbicide is demonstrated to cause selective
degeneration of dopaminergic neurons in substantia nigra
pars compacta in animal models [53]. It is known to induce
oxidative stress in mammals by participating in redox
cycling with cellular enzymes [54, 55] and in Drosophila
[56]. Data obtained in our bioassay revealed significant
reduction in the degree of paraquat-induced lethality
among flies given Cr prophylaxis clearly indicating that
flies were able to successfully mitigate the PQ induced
oxidative stress resulting in better survival rate. Further
investigations on the mechanism by which Cr protects flies
against PQ induced oxidative stress and neurotoxicty is
underway.
In conclusion, we have demonstrated the neuroprotec-
tive efficacy of creatine supplementation in Drosophila as
evidenced by its ability to modulate endogenous oxidative
markers and its propensity to mitigate rotenone induced
mitochondrial oxidative stress, restore dopamine levels and
neurotoxicty. Additional evidence of its neuroprotective
action was exemplified by the enhanced resistance to
Paraquat. While the precise mechanisms by which creatine
offers neuroprotection in this model needs further investi-
gation, we hypothesize that it may be due to the combined
effects of its antioxidative action as well as direct physio-
logical effects on mitochondria. More importantly, these
results confirm the utility of Drosophila as a primary tool
to rapidly screen compounds suspected to possess neuro-
pharmacolgical properties prior to their testing in mam-
malian models and further therapeutic use in humans.
Acknowledgments We wish to thank the Director, CFTRI for his
encouragement in this study. The first author (RKH) thanks the
University Grant Commission (UGC), New Delhi, India for the award
of Junior/Senior Research Fellowships.
Declaration of Interest The authors report no conflicts of interest.
The authors alone are responsible for the content and writing of the
manuscript.
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