Neurochem Res (2010) 35:227–238
DOI 10.1007/s11064-009-0046-1
ORIGINAL PAPER
The Effects of Polyphenols on Survival and Locomotor Activity
in Drosophila melanogaster Exposed to Iron and Paraquat
M. Jimenez-Del-Rio Æ C. Guzman-Martinez Æ
C. Velez-Pardo
Accepted: 8 August 2009 / Published online: 23 August 2009
Ó Springer Science+Business Media, LLC 2009
Abstract Parkinson’s disease (PD) is a common pro-
gressive neurodegenerative disorder, for which at present
no causal treatment is available. On the understanding that
the causes of PD are mainly oxidative stress and mito-
chondrial dysfunction, antioxidants and other drugs are
expected to be used. In the present study, we demonstrated
for the first time that pure polyphenols such as gallic acid,
ferulic acid, caffeic acid, coumaric acid, propyl gallate,
epicatechin, epigallocatechin, and epigallocatechin gallate
protect, rescue and, most importantly, restore the impaired
movement activity (i.e., climbing capability) induced by
paraquat in Drosophila melanogaster, a valid model of PD.
We also showed for the first time that high concentrations
of iron (e.g. 15 mM FeSO4) are able to diminish fly sur-
vival and movement to a similar extent as (20 mM) para-
quat treatment. Moreover, paraquat and iron synergistically
affect both survival and locomotor function. Remarkably,
propyl gallate and epigallocatechin gallate protected and
maintained movement abilities in flies co-treated with
paraquat and iron. Our findings indicate that pure poly-
phenols might be potent neuroprotective agents for the
treatment of PD against stressful stimuli.
Keywords Drosophila
Iron
Locomotor
Parkinson

Paraquat
Polyphenol
Survival
Toxicity
M. Jimenez-Del-Rio (&)
C. Guzman-Martinez

C. Velez-Pardo
School of Medicine, Medical Research Institute,
Neuroscience Research Group, University of Antioquia (UdeA),
Calle 62 # 52-59, Building 1, Room 412, Medellin,
Colombia
e-mail: marlene.jimenez@neurociencias.udea.edu.co
M. Jimenez-Del-Rio
C. Guzman-Martinez
C. Velez-Pardo
SIU, Medellin, Colombia
Introduction
Parkinson’s disease (PD) is a common progressive neu-
rodegenerative disorder characterized by the degeneration
of dopaminergic neurons in the substantia nigra zona
compacta, the decrease of the neurotransmitter dopamine
content in striatum [1] and elevated levels and/or deposits
of iron (for a review see Ref. [2]). PD is clinically
characterized by bradykinesia, rigidity, resting tremor, and
postural instability, for which at present no causal treat-
ment is available. The therapy consists only of amelio-
ration of the symptoms by replacement of deficient
dopamine (e.g. StalevoÒ). Therefore, other approaches are
critically needed for PD patients. A therapy to rescue or
protect degenerating dopamine neurons has been pro-
posed, the strategies of which should be based on the
insight of the aetiology and pathogenesis of PD. On the
understanding that the causes of PD are mainly oxidative
stress and mitochondrial dysfunction [3, 4], antioxidants,
free radical scavengers, monoamine oxidase inhibitors,
iron-chelators, and other such drugs are expected to be
used.
Substantial evidence suggests environmental risk fac-
tors such as pesticides and heavy metals as causative of
PD [5, 6]. Exposure of humans to environmental toxins
such as MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyri-
dine) and paraquat (PQ, 1,10 -dimethyl-40 ,40 -bipyridilium
dimethylsulphate) induce acute and irreversible parkin-
sonism [7–9]. Indeed, PQ2?, a nonselective herbicide, is
currently used to model PD [10] because of its similarity
to the chemical structure of the active metabolite of the
parkinsonism-induced agent MPTP, the 1-methyl-4-phe-
nylpyridium ion (MPP?). Recently, it has been shown
that paraquat is taken up across the mitochondrial inner
membrane by a carrier-mediated and membrane potential
123
228
(DWm)-dependent process and that, once in the matrix,
PQ2? is reduced to paraquat monocation radical (PQ?) by
complex I in mammalian mitochondria [11]. The PQ?
radical then rapidly reacts with oxygen (k = 7.7 9
108 M-1 s-1) to generate the superoxide anion radical
(O
-
2 ). This then sets off the well-known enzymatic or
non-enzymatic dismutation of O
- 2 into hydrogen peroxide
(H2O2), which in the presence of ferrous iron (Fe2?) is
capable of forming the highly reactive and harmful
hydroxyl radical (
OH). Therefore, the mechanism of
paraquat neurotoxicity is most likely mediated via oxi-
dative stress [12]. Interestingly, paraquat has been shown
to specifically damage dopaminergic neuronal cells in in
vivo studies with Drosophila melanogaster [13], rats [14]
and mice [15, 16]. These data clearly demonstrated that
dopaminergic neurons are responsible for the movement
function in flies and mammals and that either genetically
or environmentally induced damage of those neurons
resulted in motor dysfunction. The use of Drosophila as a
model of PD is advantageous over other models for
several reasons. First, except for cells in the gonads and
some cells in the gut, there is no cell mitosis in the adult
fly. Thus, the adult fly might be considered as an
organism of synchronously ageing cells. This character-
istic warrants precise timing at which the putative anti-
oxidant molecule impacts survival rate and/or locomotor
activity in flies. Second, Drosophila represents an
unprecedented model organism not only for understanding
fundamental neuropharmacological processes, but also for
comparative experimental research. Indeed, the similarity
between the dopaminergic network, mode of drug action,
behaviour, and gene response in D. melanogaster and
mammalian systems, has made the fly a very attractive
model for anti-parkinsonism drug discovery [17]. Third,
Drosophila offers the power of rapid drug screening
analysis, which is not yet possible in mammalian models
[18]. And finally, given the high degree of evolutionary
conservation of the human and fly genes involved in
movement disorders, Drosophila is an ideal system for
evaluating molecules with the potential of ameliorating
motor coordination [19]. Amazingly, Parkinson’s-like
motor dysfunction has been mimicked in Drosophila
either by specific genetic alterations [20–23]; pharmaco-
logical inhibition [21, 24]; or by administration of xeno-
biotic compounds such as rotenone [25] and paraquat
[13].
The study of antioxidants is becoming one of the most
important subjects in PD research. Polyphenols are a group
of chemical substances present in plants, fruits, and vege-
tables, characterized by the presence of one or more than
one phenol unit per molecule with several hydroxyl groups
on aromatic rings. Based on this structural feature, poly-
phenols are classified as phenolic acids (e.g., gallic (GA),
123
Neurochem Res (2010) 35:227–238
caffeic (CA), coumaric (CouA), ferulic acid (FA), propyl
gallate (PG), flavonoids, -the largest group of polyphenols-,
and non-flavonoid polyphenols. Flavonoids involve antho-
cyanins and anthoxantins. The latter group is divided into
flavonols, flavans, flavanols (e.g., epicatechin (EC), epi-
gallocatechin (EGC), epigallocatechin-3-gallate (EGCG)),
flavones and isoflavones [26]. Numerous studies in the past
decade have shown that polyphenols have in vitro and in
vivo activity in preventing or reducing the deleterious
effects of reactive oxygen species (ROS) associated with
oxidative stress and neurodegeneration not only because of
their strong antioxidant and metal-chelating properties [27–
29], but also because of their capability to induce intracel-
lular signalling pathways associated with cell survival and
gene expression [30, 31]. However, pro-oxidant and cell
death effects of polyphenols have also been reported [32–
34]. Thus, the question whether polyphenols are multi-
functional molecules (i.e., anti-oxidants, pro-oxidants, gene
regulators, metal-binding molecules) in vivo in humans is
still unresolved.
Several groups have studied flavonoid-rich foods rather
than pure flavonoid compounds. Therefore, little or no
clear evidence of antioxidant and chelating effects have
been demonstrated in in vivo studies. Moreover, no data
are available on whether polyphenol consumption may
improve movement alteration in PD. To establish conclu-
sive evidence for the effectiveness of polyphenols in PD, it
is essential to determine the nature of these compounds and
which of the hundreds of existing polyphenols are likely to
provide the greatest effect. Given that the fruitfly Dro-
sophila melanogaster has been proven to be a suitable
model for analyzing the interaction of genetic and envi-
ronmental factors in a PQ-induced Parkinsonism model
[13, 19], and because several of the phenolic acids and
polyphenols are natural constituents in food such as fruits,
green/black tea and white/red wine [35, 36], the aims of the
present study were (1) to assess the effect of phenolic acids
(i.e., GA, FA, CA, CouA, PG) and flavanols (i.e. EC, EGC,
EGCG) compounds (Fig. 1a) on the survival and locomo-
tor function (climbing capability) of Canton-S Drosophila
melanogaster against paraquat-toxicity, (2) to investigate
whether those compounds were able to rescue Drosophila
pre-exposed to PQ. Unquestionably, iron plays a major role
in the pathogenesis of PD [37]. However, it is yet unknown
whether iron may be a causative or secondary factor in the
disease process. We therefore investigated (3) whether iron
per se was able to induce a parkinsonism-like effect on
Drosophila and (4) whether phenolic acids and flavanols
were able to protect these flies against iron and PQ.
Understanding the mechanism of polyphenols against PQ-
induced toxicity in the Drosophila model may provide
insights into more effective antioxidant therapeutic
approaches to PD.
Neurochem Res (2010) 35:227–238
Fig. 1 a Chemical structure of polyphenols (phenolic acids and
flavanols), paraquat (inside box) and StalevoÒ (outside box) com-
pounds used in this research (and their abbreviations). b–d Schematic
Experimental Procedure
Fly Stock and Culture
Wild type Canton-S Drosophila melanogaster were main-
tained at 25°C on 12 h light/dark cycle in bottles con-
taining agar, corn meal, molasses, water, and dried yeast
medium. Propionic acid was added to prevent fungal
growth (Merck—Schuchardt OHG D-85662 Hohenbrunm
Germany) and other reagents, unless specified otherwise,
were purchased from Sigma (St. Louis, MO, USA). Female
(F) flies were collected under brief CO2 anesthesia from 2
to 3 days after eclosion.
Paraquat and Iron Toxicity Assay
The paraquat toxicity assay was performed on 2- to 3- day-old
flies collected overnight and kept on regular food medium.
Subsequently, 50 separated adult female flies were starved in
empty vials for 3 h at 25°C. Then, groups of five flies were
229
representation of feeding schedule, paraquat, iron and polyphenol
treatments in Drosophila melanogaster. For further explanation, see
‘‘Experimental Procedure’’ section
placed in ten vials containing a filter paper (Bio Rad Mini
Trans-Blot 1703932) and were pre-fed with 200 ll 55.5 mM
(1%) glucose (GLU) solution alone for 72 h. After this time,
flies were starved in empty vials for 3 h at 25°C and trans-
ferred to vials with a filter paper saturated with 20 mM
paraquat (PQ). Red food dye (8 ll/1 ml) (Red food colour
McCormick) was added to ensure homogeneity and food
intake. Living flies were counted at 6, 12, 24 and 48 h
(Fig. 1B0 ). For the iron toxicity assay, 50 separated adult
female flies were starved in empty vials for 3 h at 25°C. Then,
groups of five flies were placed in ten vials containing a filter
paper and were pre-fed with 200 ll (1%) glucose (GLU)
solution in combination with (0, 0.5, 1, 5, 10, 15, 20 mM) iron
sulphate (FeSO4) for 120 h (Fig. 1B00 ). Filters were changed
daily. Both toxicity assays were carried out by triplicate.
Antioxidant Assay
The antioxidant assay was performed on 2- to 3- day-old
female flies collected overnight and kept on regular food
123
230
medium. Subsequently, 50 females were starved in empty
vials for 3 h at 25°C. Then, groups of five F were placed in
ten vials containing a filter paper (Bio Rad Mini Trans-Blot
1703932) saturated with 200 ll fresh polyphenol solution
(gallic acid (GA), ferulic acid (FA), caffeic acid (CA),
Coumaric acid (CouA), propyl gallate (PG), epicatechin
(EC), epigallocatechin (EGC), epigallocatechin gallate
(EGCG)) at (0.1–1 mM) and 1% GLU in distilled water
(dW) for 72 h. Filters were changed daily. Then, flies were
starved in empty vials for 3 h at 25°C, and they were
placed in the vials with a filter paper saturated with either
20 mM PQ (Fig. 1C0 ), 15 mM Fe alone (Fig. 1C00 ), or in
combination with 1 mM PQ and 15 mM Fe (Fig. 1C000 ) in
1% GLU solution for different intervals of time. Red food
dye (8 ll/1 ml) (Red food colour McCormick) was added
to ensure homogeneity and food intake. Survival rate (%)
and locomotion assay were rated at each interval of time. A
total number of 150 flies were used for each substance.
Rescue Assay
The rescue assay was performed on 2- to 3-d-old flies
collected overnight and kept on regular food medium.
Groups of five F flies were placed in plastic vials con-
taining a filter paper saturated with 1% GLU in dW for
72 h. Filters were changed daily. Then, flies were starved
in empty vials for 3 h at 25°C, and transferred to vials
containing a filter paper saturated with 20 mM PQ and 1%
GLU in dW for 6, 12 and 24 h, respectively at 25°C. Then,
50 female flies from each interval of time were placed in
groups of five in ten plastic vials with a filter paper satu-
rated with 0.1–0.5 mM polyphenols with 1% GLU in dW
for an additional 24 h, i.e. survival rate (%) and locomotion
assay were recorded at 30, 36, and 48 h (Fig. 1d). The
rescue assay was carried out by triplicate.
Locomotion Assay
The movement deficits assay was performed on treated
flies according to ref. [13] with minor modifications.
Briefly, treated female flies were placed in empty plastic
vials. After a 10 min rest period, the flies were tapped to
the bottom of the vials, and the number of flies able to
climb 5 cm in 6 s was recorded at each interval of time.
The assays were repeated three times at 1 min intervals.
The scores are the mean of the numbers of flies at the top
(ntop) and at the bottom (nbot), expressed as percentages of
the total number of flies (ntot). Results are presented as the
mean ± SD of the scores obtained in three independent
experiments. For each experiment, a performance index
(PI) was calculated, defined as 1/2[(ntot ? ntop - nbot)/ntot]
[25]. Statistical analysis was performed on the PIs with the
Student’s t test. StalevoÒ (carbidopa, levodopa and
123
Neurochem Res (2010) 35:227–238
entacapone), a drug amply medicated for the treatment of
Parkinson’s disease, was used as a positive restorative drug
against PQ toxicity.
Statistical Analysis
Data were shown as a mean ± standard deviation of the
mean (SD) for three independent experiments. The Chi
Square statistic was performed to compare proportion of
percentage between independent groups. Differences were
considered statistically significant at P \ 0.05.
Results
Polyphenols Protected and Maintained the Locomotion
Function of Drosophila melanogaster Exposed to
Paraquat
Previously, we have shown that 20 mM paraquat signifi-
cantly reduces survival and diminishes locomotor activity in
Drosophila melanogaster [19]. To evaluate whether poly-
phenols (Fig. 1a) were able to protect and to maintain the
locomotion function of D. melanogaster against paraquat,
flies were pre-fed with different concentrations of poly-
phenols for 72 h, and then fed with 20 mM PQ for 24 and
48 h. Taken as a whole, phenolic acids and flavanols were
able to protect D. melanogaster against paraquat at the
concentrations tested and time of exposure (Fig. 2a–c).
Interestingly, low concentrations (0.1 mM, Fig. 2a) of
polyphenols, (except CA (1 mM)), were more effective than
medium (0.5 mM, Fig. 2b) or high (1 mM, Fig. 2c) con-
centrations to protect the specimens from PQ noxious effects
at 24 h. Likewise, except for GA and CouA, polyphenols
such as PG (0.1 mM), FA (0.5 mM), CA (0.5 mM) and
catechins (EC, 0.5 mM; EGC, 0.1 mM; EGCG, 0.1 mM)
protected against PQ exposure at 48 h. It is worth men-
tioning that flies pre-fed with different concentrations of
polyphenols for 72 h, and then fed with 1% GLU for 24 and
48 h (controls) showed survival rate and climbing percent-
age values similar to flies fed with just 1% GLU for 120 h.
Remarkably, 0.5 mg/ml StalevoÒ (equivalent to 0.76 mM
levodopa, 0.17 mM cardidopa and 1 mM entacapone), a
drug used in the treatment of PD, offered D. melanogaster
100 and 26% survival rate when exposed to PQ for 24 and
48 h, respectively. Nevertheless, 0.1 and 1 mg/ml StalevoÒ
significantly increased the flies’ survival percentage com-
pared to flies treated with PQ alone for the same time points.
We then evaluated whether polyphenols were able to
maintain locomotor functionality in D. melanogaster
exposed to PQ. Therefore, flies were pre-fed with the
polyphenol concentration at which the maximal survival
percentage was observed. i.e., 0.1 mM GA, FA, CouA, PG,
Neurochem Res (2010) 35:227–238
Fig. 2 Protective effect of polyphenols in D. melanogaster exposed
to paraquat. Female flies were pre-fed with either 1% glucose alone,
0.1 mM (a), 0.5 mM (b), or 1 mM (c) gallic acid (GA), ferulic acid
(FA), caffeic acid (CA), coumaric acid (CouA), propyl gallate (PG),
epicatechin (EC), epigallocatechin (EGC), epigallocatechin gallate
(EGCG) polyphenols and 0.1–1 mg/ml StalevoÒ with 1% glucose in
EC EGC, EGCG; 0.5 mM CA, and as a positive control,
0.5 mg/ml StalevoÒ for 72 h. After this time, flies were
treated with PQ and locomotor assessment was performed
at different time points. As shown in Fig. 2d, the flies’
locomotor activity (%) is significantly reduced by 13, 56,
77, and 88% at 6, 12, 24 and 48 h, respectively under PQ
only (negative control), whereas those treated with the
polyphenols were able to maintain their climbing ability.
The restorative effect of all polyphenols began to be effec-
tive after 6 h post-PQ exposure. In fact, negative geotaxis
movement increased by 46–97% at 12 h, 143–334% at
24 h, 333–600% at 48 h, respectively post-PQ treatment.
Strikingly, StalevoÒ showed climbing capability results
comparable to polyphenols at the different time point
evaluations.
Polyphenols Rescued and Restored Locomotive
Function in D. melanogaster Flies from Paraquat
Intoxication
To evaluate whether polyphenols were able to rescue
D. melanogaster against PQ-induced toxicity and
231
distilled water (dW) for 72 h. Then, flies were left untreated (U) or
treated with 20 mM paraquat (PQ) for 24 and 48 h. For locomotion
assay, 0.1 mM polyphenol concentration was used. a–c Survival rate
(%) and d locomotion assay were recorded at indicated time.
Polyphenols were statistically significant (** P \ 0.001) vs. PQ at
24 h. * P \ 0.05, ** P \ 0.001 polyphenols vs. PQ at 48 h
movement impairment, the flies were pre-fed with 1%
GLU for 72 h, then exposed to PQ for 6, 12, and 24 h. At
each period of time, flies were fed with 1% GLU (control)
or polyphenols for an additional 24 h (Fig. 1d). As illus-
trated in Fig. 3a, polyphenols were effective rescuing
D. melanogaster with variable efficiencies. While CouA,
PG, EC, and EGCG significantly rescued D. melanogaster
from the toxic effect of PQ, FA showed no effect, but GA
and CA were effective at 48 h post-PQ treatment.
Noticeably, polyphenols were able to maintain and restore
the flies’ movement abilities (Fig. 3b). StalevoÒ showed
comparable results to polyphenols at different time points.
Polyphenols Protected and Restored Locomotive
Function in D. melanogaster Flies from Iron and
Paraquat Exposure
It is well known that iron plays an important role in the
pathophysiology of PD [2]. To evaluate whether iron was
toxic or whether it affected movement in D. melanogaster,
they were fed with increasing concentrations of iron sul-
phate (0.5–20 mM, FeSO4) for up to 120 h (Fig. 1b). As
123
232 Neurochem Res (2010) 35:227–238

Fig. 3 Rescue effect of

polyphenols in D. melanogaster
exposed to paraquat. Female
flies were pre-fed with 1% GLU
solution for 72 h. After this
time, a group of flies were
exposed to either 1% GLU
alone (GLU) or 20 mM PQ and
1% GLU in dW for 6, 12, and
24 h (control group). After each
time interval, a group of flies
were fed with 1% GLU solution
(U), or with 0.1 mM gallic acid
(GA), ferulic acid (FA),
coumaric acid (CouA), propyl
gallate (PG), epicatechin (EC),
epigallocatechin (EGC),
epigallocatechin gallate
(EGCG), 0.5 mM caffeic acid
(CA) polyphenols and 0.5 mg/
ml StalevoÒ for an additional
24 h. a Survival rate (%) and b
locomotion assay were recorded
at the indicated time.
* P \ 0.05 polyphenols vs. PQ,
** P \ 0.001 polyphenols vs.
PQ

shown in Fig. 4, iron induced a significant reduction in
survival and locomotor function in a concentration and
time dependent fashion. In fact, while 5 mM iron dimin-
ished survival by 40% at 96 h and reduced locomotor
abilities by 50% at 120 h (Fig. 4b), higher concentrations
of iron were moderately toxic (10–15 mM) or completely
lethal (20 mM) to Drosophila (Fig. 4a). Given that 15 mM
Fe reduces the flies’ survival by 60–90% and impairs
movement (i.e., climbing abilities) by 50–90% at 72 and
96 h, respectively, this concentration was chosen for fur-
ther experiments. Moreover, this concentration induced a
comparable effect on survival and climbing alterations as
did 20 mM PQ at 48 h.
To further determine whether polyphenols were able to
protect D. melanogaster against the harmful effects of iron,
we fed flies with phenolic acid (e.g. GA, PG) and flavanols
(e.g., EC, EGC, EGCG) with 15 mM iron for 120 h
(Fig. 1C00 ). As shown in Fig. 5a, GA and PG were more
efficient protecting flies by 100% and 450% survival at 72
and 96 h, respectively than flavanols. Similarly, GA and
PG maintained movement capabilities higher than EC,
EGC, EGCG at 48 and 72 h, respectively (Fig. 5b).
Noticeably, although polyphenols were able to prolong
survival for up to 120 h, they were unable to restore
movement at this period of time.
To evaluate whether polyphenols may protect Dro-
sophila against iron and PQ harmful effects, we pre-fed
flies with PG and EGCG for 72 h, then flies were fed with
1 mM PQ, 15 mM FeSO4 or with PQ and FeSO4 for
additional 72 h (Fig. 1C000 ). Interestingly, both PG and
EGCG polyphenols increased Drosophila’s survival and
climbing abilities when treated with iron or PQ alone, or
iron in combination with paraquat up to 120 h (Figs. 6a, b
and 7a, b). As expected, PQ and iron caused 100% death in
Drosophila by the second day of treatment (data not
shown).

123
Neurochem Res (2010) 35:227–238
Fig. 4 Effect of iron in D. melanogaster. Female flies were pre-fed with either 1% glucose alone, or with 0.1–20 mM iron with 1% glucose in
distilled water (dW) for 120 h. a Survival rate (%) and b locomotion assay were recorded at the indicated time
Discussion
It is known that the mechanism of paraquat toxicity in
dopaminergic neurons is most likely mediated via oxida-
tive stress [12]. Indeed, paraquat (PQ2?) generates super-
oxide radicals (O
- 2 ) by undergoing a NADH-dependent
reduction to form a stable paraquat monocation (PQ?)
radical that reacts very rapidly (k2 = 7.7 9 108 M s-1)
with O2 to generate PQ2? and O
- 2 radicals [38], which in
turn produces H2O2 by either enzymatic or non-enzymatic
dismutation reaction. Therefore, any molecule or com-
pound capable of blocking either O
- 2 radicals or H2O2
generation might have potential antioxidant capacities [39].
In this regard, polyphenols are the most abundant antiox-
idants in our diet [36]. To establish conclusive evidence for
the effectiveness of polyphenols in neurodegenerative
233
disorders, it is therefore crucial to determine which of the
hundreds of existing polyphenols are likely to provide the
greatest effect. Since phenolic acids and flavanols present
in fruits, white/red wine and green/black tea [36] are the
most extensively studied in in vivo and in vitro assays and
are currently considered to be of great nutritional interest,
we have selected some of the most representative poly-
phenols (Fig. 1a) for our experimental settings.
In the present study, we demonstrated for the first time
that pure polyphenols such as GA, FA, CA, CouA, PG, EC,
EGC and EGCG protect, rescue and most importantly,
restore the impaired movement activity (i.e. climbing
capability) induced by PQ in Drosophila melanogaster, a
valid model of PD [19]. We also showed for the first time
that high concentrations of iron (sulphate) are able to
diminish fly survival and movement to a similar extent as
123
234
Fig. 5 Protective effect of polyphenols in D. melanogaster exposed
to iron. Female flies were fed with either 1% glucose alone, 0.1 mM
gallic acid (GA), propyl gallate (PG), epicatechin (EC), epigalloca-
techin (EGC), epigallocatechin gallate (EGCG) polyphenols or
PQ treatment. Moreover, PQ and iron synergistically affect
both survival and locomotor function. Remarkably, PG and
EGCG polyphenols protected and maintained movement
abilities in flies co-treated with PQ/iron. Specifically, we
found that 0.1–1 mM polyphenols protect and rescue
Drosophila against 20 mM PQ. A study similar to ours has
been performed by Kim and colleagues [40], but with
important differences in study design and findings. In
that study, wild type isogenic Drosophila Canton S flies
(100–150/bottle) were pre-fed with (?)-catechin and (-)-
epicatechin (50 mg/ml equivalent to 167 lM) for 24 h, and
transferred to bottles containing both paraquat (5 mM) and
the antioxidant. The survival percentage was checked after
48 and 96 h. They found no preventive effect against PQ
123
Neurochem Res (2010) 35:227–238
15 mM iron. a Survival rate (%) and b locomotion assay were
recorded at the indicated time. * P \ 0.05 vs. control (iron alone),
** P \ 0.001 vs. control (iron alone)
toxicity. The authors offered no explanation for these
findings. Based on our survival test results, a more likely
explanation for their failure to find a protective effect of an
antioxidant effect is because the duration of antioxidant
feeding was too short and the antioxidant concentration
was too low in their study to observe the full enhancing
effect of polyphenols on survival rate. It should be noted
that implicit in the design of our study is the assumption
that polyphenols are antioxidants, however, in vitro liter-
ature suggest that they may be pro-oxidant at certain con-
centrations [32–34], but our data, and those of others,
suggest that over a very wide dose range this is not the case
in vivo [41, 42]. Although we do not disregard that poly-
phenol supplementation, especially catechin derivatives
Neurochem Res (2010) 35:227–238 235

Fig. 6 Protective effect of

propyl gallate in
D. melanogaster exposed to iron
and paraquat. Female flies were
pre-fed with 0.1 mM PG and
1% GLU solution for 72 h.
After this time, a group of flies
were exposed to either 1% GLU
alone (GLU), 15 mM iron,
1 mM PQ, or 15 mM iron/
1 mM PQ and 1% GLU in dW
for an additional 72 h. a
Survival rate (%) and b
locomotion assay were recorded
at the indicated time with
respect to iron (control).
* P \ 0.05, ** P \ 0.001

EC, ECG, EGCG could increase survival time of D. mel-
anogaster under PQ by other non-antioxidant mechanisms
e.g., by up-regulating the enzymatic activity of CuZnSOD,
MnSOD and catalase with reduction of lipid peroxidation
[41], our data suggest that the antioxidant activity of
polyphenols (i.e., phenolic acids and flavanols) can be
attributed to its unique phenolic structure, which can
donate an electron or proton to a free radical. In support of
this view, it has been demonstrated that GA, CA, FA, EC,
EGC, EGCG [43] are able to reduce the free radical DPPH

according to a DPPH
reduction test. Furthermore, phenolic
acids have been shown to scavenge H2O2 [44]. In accor-
dance with these data, we have recently found that PG, EC
and EGCG effectively attenuate mitochondrial damage
against PQ-induced oxidative stress by scavenging O
-
radical and H2O2 thereby maintaining mitochondrial
functionality by keeping high DWm (M. Jimenez-Del-Rio,
C. Velez-Pardo, J. Bustamante, S. Lores-Arnaiz, Unpub-
lished observations). Taken together our data comply with
the notion that the antioxidant capacity of polyphenols
against paraquat is dependent on ROS scavenger activity
and mitochondrial protection.
The most interesting finding in the present study was
that phenolic acids and flavanol supplementation was also
associated with a significant recovery of Drosophila’s
locomotion activity impaired by PQ. Although, times of
effectiveness of (0.1 mM) polyphenols differ from one to
another, the sequence of their efficacy against PQ treatment
was characterized as follows (for comparative purpose,
StalevoÒ was included): FA=EGCG[ EGC[ PG=CouA=
EC= StalevoÒ[ GA= CA at 12 h; GA[ CA[ EGCG=
2 EC[EGC= StalevoÒ[ PG[ FA= CouA at 24 h; and
CouA[ PG= FA=EGCG[CA= GA= EGC= StalevoÒ[ EC
at 48 h. These data show that polyphenols positively affect

123
236 Neurochem Res (2010) 35:227–238

Fig. 7 Protective effect of

epigallocatechin gallate in
D. melanogaster exposed to iron
and paraquat. Female flies were
pre-fed with 0.1 mM EGCG and
1% GLU solution for 72 h.
After this time, a group of flies
were exposed to either 1% GLU
alone (GLU), 15 mM iron,
1 mM PQ, or 15 mM iron/
1 mM PQ and 1% GLU in dW
for an additional 72 h. a
Survival rate (%) and b
locomotion assay were recorded
at the indicated time with
respect to iron (control).
* P \ 0.05, ** P \ 0.001

flies’ movement, thus the restorative negative geotaxis
movement increased overall by 46–97% at 12 h, 143–334%
at 24 h, 333–600% at 48 h, post-PQ treatment. Taken
together these data suggest that polyphenols are efficient
compounds bringing-back Drosophila melanogaster to
normal movement functionality. Noticeably, polyphenols
were even more effective than StalevoÒ, a currently used
medicine to treat patients with PD. Indeed, StalevoÒ con-
tains three active substances involved in the metabolism of
dopamine: levodopa, which acts as a dopamine precursor;
carbidopa, which is dopa decarboxylase inhibitor and ent-
acapone, which is a catechol-O-methyl transferase inhibitor
(Fig. 1a). Interestingly, these components are structurally
similar to polyphenols. Although dopa decarboxylase and
methyl transferase enzymes are expressed in D. melano-
gaster [45], we do not discard the possibility that the
protective, rescue and locomotor activity of StalevoÒ was
associated, at least in part, to antioxidant activity of its
components. However, the lack of polyphenols and Sta-
levoÒ bioavailability data in D. melanogaster discouraged
us from concluding that a similar effect of movement
amelioration in PD patients might be observed with poly-
phenol intake. Unfortunately, at present no data is available
on studies within the effects of pure polyphenols in PD
patients with emphasis in locomotor assessment. Further
studies are needed to clarify this issue.
Recently, it has been shown that binding of the poly-
phenol to iron is essential for antioxidant activity [46]. On
the other hand, increasing amounts of data suggest that iron
might be involved in the pathogenesis of PD [37]. Indeed,
this metal has been found to accumulate in individual
substancia nigra dopaminergic neurons from unfixed frozen
post-mortem tissue of PD patients [47] and in substancia
nigra from PD patients [48]. However, it is yet unknown
whether iron may be a causative or secondary factor in the
disease process. Therefore, we initially investigated whe-
ther iron per se was able to induce parkinsonism-like effect
on Drosophila. In the present investigation, we have shown
for the first time that feeding Drosophila with moderately
high concentrations of iron (5–15 mM) for up to 120 h

123
Neurochem Res (2010) 35:227–238
effectively affects flies’ negative geotaxis movements,
albeit with a relative decrease on survival rate. This result
suggests that iron, at least under experimental conditions,
might be able to target dopaminergic neurons in the fly.
Several observations support this view. First, proteins
involved in iron uptake, transport and storage in D. mela-
nogaster are highly homologous to those proteins involved
in the metabolism of iron in humans [49]. Second, dopa-
minergic neurons in D. melanogaster are vulnerable in a
similar manner as the human dopaminergic neurons to
oxidative stress and cell death induced by xenobiotic
compound exposure in in vivo models such as paraquat
[13], rotenone [25] and iron [50–52]. One possible expla-
nation for this sensitivity could be related to the fact that
iron accelerates dopamine oxidation, thereby producing
reactive oxygen species such as H2O2 which in turn react
with Fe2? ions generating hydroxyl radicals (
OH), a more
reactive molecule towards proteins, RNA and DNA [53].
However, further studies are needed to specifically evi-
dence deposition of iron and damage of the dopaminergic
neurons in Drosophila. Taken together our data suggest
that iron could be a potential environmental causative
factor of sporadic PD.
Another goal of our investigation was to establish
whether polyphenols were able to protect flies against iron
and PQ. As expected, co-supplementation of (1 mM)
paraquat and (15 mM) iron was extremely toxic to
D. melanogaster [40]. However, the phenolic acid PG and
flavanols EGCG efficiently protected Drosophila exposed
either to iron alone or in combination with iron and para-
quat. Similar survival rates were observed with GA and
EGC (data not shown). These data comply with the notion
that iron exacerbates paraquat-induced toxicity in in vivo
[54], but the capability of polyphenols to form iron-com-
plexes and to scavenge free radicals might protect dopa-
minergic neurons from PQ-induced O
- 2 and H2O2 harmful
effects (e.g., Fenton reaction). Consequently, dopaminergic
neurons might conserve their functionality. Indeed, in the
present investigation we report for the first time that
polyphenols were able to maintain locomotor function in
D. melanogaster supplemented with paraquat, iron or a
combination of iron and paraquat. These findings suggest
that pre-treatment with polyphenols might be helpful in
reducing iron and paraquat-induced toxicity and movement
impairment in adult Drosophila.
Despite the fact that the multifunctional activities of
polyphenols (i.e., antioxidants, pro-oxidants, gene regula-
tors, metal-binding molecules) have not yet been clearly
established to occur in vivo in humans, and bioavailability
data of polyphenols in vivo in PD patients is needed, the
results of this study should provide a framework for future
studies to assess potential antioxidant capacity of new
polyphenols and/or polyphenol-related structural molecules
237
to suppress oxidative stress and to restore and/or maintain
locomotor activity in PD patients.
Acknowledgments This work was supported by Colciencias grants
#1115-408-20504, and CODI-U.deA. grants #2408 awarded to
C.V.-P. and M.J.-Del-Rio.
References
1. Forno LS (1996) Neuropathology of Parkinson’s disease. J
Neuropathol Exp Neurol 55:259–272
2. Berg D (2007) Disturbance of iron metabolism as a contributing
factor to SN hyperechogenicity in Parkinson’s disease: implica-
tions for idiopathic and monogenetic forms. Neurochem Res
32:1646–1654
3. Henchcliffe C, Beal MF (2008) Mitochondrial biology and oxi-
dative stress in Parkinson disease pathogenesis. Nat Clin Pract
Neurol 4:600–609
4. Zhou C, Huang Y, Przedborski S (2008) Oxidative stress in
Parkinson’s disease: a mechanism of pathogenic and therapeutic
significance. Ann N Y Acad Sci 1147:93–104
5. Dick FD, De Palma G, Ahmadi A, Scott NW, Prescott GJ,
Bennett J, Semple S, Dick S, Counsell C, Mozzoni P, Haites N,
Wettinger SB, Mutti A, Otelea M, Seaton A, Söderkvist P, Felice
A, Geoparkinson study group (2007) Environmental risk factors
for Parkinson’s disease and parkinsonism: the Geoparkinson
study. Occup Environ Med 64:666–672
6. Jones DC, Miller GW (2008) The effects of environmental neu-
rotoxicants on the dopaminergic system: a possible role in drug
addiction. Biochem Pharmacol 76:569–581
7. Ballard PA, Tetrud JW, Langston JW (1985) Permanent human
parkinsonism due to 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyr-
idine (MPTP): seven cases. Neurology 35:949–956
8. Hertzman C, Wiens M, Bowering D, Snow B, Calne D (1990)
Parkinson’s disease: a case–control study of occupational and
environmental risk factors. Am J Ind Med 17:349–355
9. Liou HH, Tsai MC, Chen CJ, Jeng JS, Chang YC, Chen SY, Chen
RC (1997) Environmental risk factors and Parkinson’s disease: a
case–control study in Taiwan. Neurology 48:1583–1588
10. Bové J, Prou D, Perier C, Przedborski S (2005) Toxin-induced
models of Parkinson’s disease. NeuroRx 2:484–894
11. Cochemé HM, Murphy MP (2008) Complex I is the major site of
mitochondrial superoxide production by paraquat. J Biol Chem
283:1786–1798
12. Dinis-Oliveira RJ, Remiao F, Carmo H, Duarte JA, Navarro AS,
Bastos ML, Carvalho F (2006) Paraquat exposure as an etiolog-
ical factor of Parkinson’s disease. Neurotoxicology 27:1110–
1122
13. Chaudhuri A, Bowling K, Funderburk C, Lawal H, Inamdar A,
Wang Z, O’Donnell JM (2007) Interaction of genetic and envi-
ronmental factors in a Drosophila parkinsonism model. J Neu-
rosci 27:2457–2467
14. Kuter K, Smiałowska M, Wierońska J, Zieba B, Wardas J, Pie-
traszek M, Nowak P, Biedka I, Roczniak W, Konieczny J,
Wolfarth S, Ossowska K (2007) Toxic influence of subchronic
paraquat administration on dopaminergic neurons in rats. Brain
Res 1155:196–207
15. Li X, Yin J, Cheng CM, Sun JL, Li Z, Wu YL (2005) Paraquat
induces selective dopaminergic nigrostriatal degeneration in
aging C57BL/6 mice. Chin Med J (Engl) 118:1357–1361
16. Prasad K, Tarasewicz E, Mathew J, Strickland PA, Buckley B,
Richardson JR, Richfield EK (2009) Toxicokinetics and
123
238
toxicodynamics of paraquat accumulation in mouse brain. Exp
Neurol 215:358–367
17. Nichols CD (2006) Drosophila melanogaster neurobiology,
neuropharmacology, and how the fly can inform central nervous
system drug discovery. Pharmacol Ther 112:677–700
18. Manev H, Dimitrijevic N, Dzitoyeva S (2003) Techniques: fruit
flies as models for neuropharmacological research. Trends
Pharmacol Sci 24:41–43
19. Jimenez-Del-Rio M, Daza-Restrepo A, Velez-Pardo C (2008)
The cannabinoid CP55, 940 prolongs survival and improves
locomotor activity in Drosophila melanogaster against paraquat:
implications in Parkinson’s disease. Neurosci Res 61:404–411
20. Feany MB, Bender WW (2000) A Drosophila model of Parkin-
son’s disease. Nature 404:394–398
21. Pendleton RG, Rasheed A, Sardina T, Tully T, Hillman R (2002)
Effects of tyrosine hydroxylase mutants on locomotor activity in
Drosophila: a study in functional genomics. Behav Genet 32:
89–94
22. Wang C, Lu R, Ouyang X, Ho MW, Chia W, Yu F, Lim KL
(2007) Drosophila overexpressing parkin R275W mutant exhibits
dopaminergic neuron degeneration and mitochondrial abnormal-
ities. J Neurosci 27:8563–8570
23. Sang TK, Chang HY, Lawless GM, Ratnaparkhi A, Mee L,
Ackerson LC et al (2007) A Drosophila model of mutant human
parkin-induced toxicity demonstrates selective loss of dopami-
nergic neurons and dependence on cellular dopamine. J Neurosci
27:981–992
24. Pendleton RG, Parvez F, Sayed M, Hillman R (2002) Effects of
pharmacological agents upon a transgenic model of Parkinson’s
disease in Drosophila melanogaster. J Pharmacol Exp Ther
300:91–96
25. Coulom H, Birman S (2004) Chronic exposure to rotenone
models sporadic Parkinson’s disease in Drosophila melanogaster.
J Neurosci 24:10993–10998
26. D’Archivio M, Filesi C, Di Benedetto R, Gargiulo R, Giovannini
C, Masella R (2007) Polyphenols, dietary sources and bioavail-
ability. Ann Ist Super Sanita 43:348–361
27. Sestili P, Diamantini G, Bedini A, Cerioni L, Tommasini I, Tarzia
G, Cantoni O (2002) Plant-derived phenolic compounds prevent
the DNA single-strand breakage and cytotoxicity induced by tert-
butylhydroperoxide via an iron-chelating mechanism. Biochem J
364(Pt 1):121–128
28. Melidou M, Riganakos K, Galaris D (2005) Protection against
nuclear DNA damage offered by flavonoids in cells exposed to
hydrogen peroxide: the role of iron chelation. Free Radic Biol
Med 39:1591–1600
29. Perron NR, Brumaghim JL (2009) A review of the antioxidant
mechanisms of polyphenol compounds related to iron binding.
Cell Biochem Biophys 53:75–100
30. Ramassamy C (2006) Emerging role of polyphenolic compounds
in the treatment of neurodegenerative diseases: a review of their
intracellular targets. Eur J Pharmacol 545:51–64
31. Zaveri NT (2006) Green tea and its polyphenolic catechins:
medicinal uses in cancer and noncancer applications. Life Sci
78:2073–2080
32. Galati G, Sabzevari O, Wilson JX, O’Brien PJ (2002) Prooxidant
activity and cellular effects of the phenoxyl radicals of dietary
flavonoids and other polyphenolics. Toxicology 177:91–104
33. Elbling L, Weiss RM, Teufelhofer O, Uhl M, Knasmueller S,
Schulte-Hermann R, Berger W, Micksche M (2005) Green tea
extract and (-)-epigallocatechin-3-gallate, the major tea catechin,
exert oxidant but lack antioxidant activities. FASEB J 19:
807–809
34. Shin JK, Kim GN, Jang HD (2007) Antioxidant and pro-oxidant
effects of green tea extracts in oxygen radical absorbance
capacity assay. J Med Food 10:32–40
123
Neurochem Res (2010) 35:227–238
35. Beecher GR (2003) Overview of dietary flavonoids: nomencla-
ture, occurrence and intake. J Nutr 133:3248S–3254S
36. USDA (2007) Database for the flavonoid content of selected
foods. Release 2.1 January 2007. http://www.ars.usda.gov/
nutrientdata (available on August 2009)
37. Berg D, Youdim MB (2006) Role of iron in neurodegenerative
disorders. Top Magn Reson Imaging 17:5–17
38. Farrington JA, Ebert M, Land EJ, Fletcher K (1973) Bipyridylium
quaternary salts and related compounds. V. Pulse radiolysis
studies of the reaction of paraquat radical with oxygen. Impli-
cations for the mode of action of bipyridyl herbicides. Biochim
Biophys Acta 314:372–381
39. Huang D, Ou B, Prior RL (2005) The chemistry behind antiox-
idant capacity assays. J Agric Food Chem 53:1841–1856
40. Kim SJ, Han D, Ahn BH, Rhee JS (1997) Effect of glutathione,
catechin, and epicatechin on the survival of Drosophila mela-
nogaster under paraquat treatment. Biosci Biotechnol Biochem
61:225–229
41. Li YM, Chan HY, Huang Y, Chen ZY (2007) Green tea catechins
upregulate superoxide dismutase and catalase in fruit flies. Mol
Nutr Food Res 51:546–554
42. Liu H, Guo Z, Xu L, Hsu S (2008) Protective effect of green tea
polyphenols on tributyltin-induced oxidative damage detected by
in vivo and in vitro models. Environ Toxicol 23:77–83
43. Villaño D, Fernandez-Pachon MS, Moya ML, Troncoso AM,
Garcia-Parrila MC (2007) Radical scavenging ability of poly-
phenolic compounds towards DPPH free radical. Talanta 71:
230–235
44. Sroka Z, Cisowski W (2003) Hydrogen peroxide scavenging,
antioxidant and anti-radical activity of some phenolic acids. Food
Chem Toxicol 41:753–758
45. Beall CJ, Hirsh J (1987) Regulation of the Drosophila dopa
decarboxylase gene in neuronal and glial cells. Genes Dev 1:
510–520
46. Perron NR, Hodges JN, Jenkins M, Brumaghim JL (2008) Pre-
dicting how polyphenol antioxidants prevent DNA damage by
binding to iron. Inorg Chem 47:6153–6161
47. Oakley AE, Collingwood JF, Dobson J, Love G, Perrott HR,
Edwardson JA, Elstner M, Morris CM (2007) Individual dopa-
minergic neurons show raised iron levels in Parkinson disease.
Neurology 68:1820–1825
48. Wallis LI, Paley MN, Graham JM, Grünewald RA, Wignall EL,
Joy HM, Griffiths PD (2008) MRI assessment of basal ganglia
iron deposition in Parkinson’s disease. J Magn Reson Imaging
28:1061–1067
49. Nichol H, Law JH, Winzerling JJ (2002) Iron metabolism in
insects. Annu Rev Entomol 47:535–559
50. Ben-Shachar D, Youdim MB (1991) Intranigral iron injection
induces behavioral and biochemical ‘‘parkinsonism’’ in rats. J
Neurochem 57:2133–2135
51. Sengstock GJ, Olanow CW, Menzies RA, Dunn AJ, Arendash
GW (1993) Infusion of iron into the rat substantia nigra: nigral
pathology and dose–dependent loss of striatal dopaminergic
markers. J Neurosci Res 35:67–82
52. Sengstock GJ, Olanow CW, Dunn AJ, Barone S Jr, Arendash GW
(1994) Progressive changes in striatal dopaminergic markers,
nigral volume, and rotational behavior following iron infusion
into the rat substantia nigra. Exp Neurol 130:82–94
53. Hattoria N, Wanga M, Taka H, Fujimura T, Yoritaka A, Kubo S,
Mochizuki H (2009) Toxic effects of dopamine metabolism in
Parkinson’s disease. Parkinsonism Relat Disord 15(Suppl 1):
S35–S38
54. Peng J, Peng L, Stevenson FF, Doctrow SR, Andersen JK (2007)
Iron and paraquat as synergistic environmental risk factors in
sporadic Parkinson’s disease accelerate age-related neurodegen-
eration. J Neurosci 27:6914–6922