The Effects of Polyphenols on Survival and Locomotor Activity in Drosophila melanogaster Exposed to Iron and Paraquat
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- Jimenez-Del-Rio, M., Guzman-Martinez, C. & Velez-Pardo, C. Neurochem Res (2010) 35: 227. doi:10.1007/s11064-009-0046-1
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Parkinson’s disease (PD) is a common progressive neurodegenerative disorder, for which at present no causal treatment is available. On the understanding that the causes of PD are mainly oxidative stress and mitochondrial 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 survival and movement to a similar extent as (20 mM) paraquat 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 polyphenols might be potent neuroprotective agents for the treatment of PD against stressful stimuli.
Parkinson’s disease (PD) is a common progressive neurodegenerative disorder characterized by the degeneration of dopaminergic neurons in the substantia nigra zona compacta, the decrease of the neurotransmitter dopamine content in striatum  and elevated levels and/or deposits of iron (for a review see Ref. ). PD is clinically characterized by bradykinesia, rigidity, resting tremor, and postural instability, for which at present no causal treatment is available. The therapy consists only of amelioration of the symptoms by replacement of deficient dopamine (e.g. Stalevo®). Therefore, other approaches are critically needed for PD patients. A therapy to rescue or protect degenerating dopamine neurons has been proposed, 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 factors 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-tetrahydropyridine) and paraquat (PQ, 1,1′-dimethyl-4′,4′-bipyridilium dimethylsulphate) induce acute and irreversible parkinsonism [7–9]. Indeed, PQ2+, a nonselective herbicide, is currently used to model PD  because of its similarity to the chemical structure of the active metabolite of the parkinsonism-induced agent MPTP, the 1-methyl-4-phenylpyridium ion (MPP+). Recently, it has been shown that paraquat is taken up across the mitochondrial inner membrane by a carrier-mediated and membrane potential (ΔΨm)-dependent process and that, once in the matrix, PQ2+ is reduced to paraquat monocation radical (PQ+) by complex I in mammalian mitochondria . The PQ+ radical then rapidly reacts with oxygen (k = 7.7 × 108 M−1 s−1) to generate the superoxide anion radical (O2·−). This then sets off the well-known enzymatic or non-enzymatic dismutation of O2·− 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 oxidative stress . Interestingly, paraquat has been shown to specifically damage dopaminergic neuronal cells in in vivo studies with Drosophila melanogaster , rats  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 characteristic warrants precise timing at which the putative antioxidant 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 . Third, Drosophila offers the power of rapid drug screening analysis, which is not yet possible in mammalian models . 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 . Amazingly, Parkinson’s-like motor dysfunction has been mimicked in Drosophila either by specific genetic alterations [20–23]; pharmacological inhibition [21, 24]; or by administration of xenobiotic compounds such as rotenone  and paraquat .
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 vegetables, 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, polyphenols are classified as phenolic acids (e.g., gallic (GA), caffeic (CA), coumaric (CouA), ferulic acid (FA), propyl gallate (PG), flavonoids, -the largest group of polyphenols-, and non-flavonoid polyphenols. Flavonoids involve anthocyanins and anthoxantins. The latter group is divided into flavonols, flavans, flavanols (e.g., epicatechin (EC), epigallocatechin (EGC), epigallocatechin-3-gallate (EGCG)), flavones and isoflavones . 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 intracellular 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 multifunctional molecules (i.e., anti-oxidants, pro-oxidants, gene regulators, metal-binding molecules) in vivo in humans is still unresolved.
Fly Stock and Culture
Wild type Canton-S Drosophila melanogaster were maintained at 25°C on 12 h light/dark cycle in bottles containing 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 placed in ten vials containing a filter paper (Bio Rad Mini Trans-Blot 1703932) and were pre-fed with 200 μl 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 transferred to vials with a filter paper saturated with 20 mM paraquat (PQ). Red food dye (8 μl/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. 1B′). 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 μl (1%) glucose (GLU) solution in combination with (0, 0.5, 1, 5, 10, 15, 20 mM) iron sulphate (FeSO4) for 120 h (Fig. 1B″). Filters were changed daily. Both toxicity assays were carried out by triplicate.
The antioxidant assay was performed on 2- to 3- day-old female flies collected overnight and kept on regular food 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 μl 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. 1C′), 15 mM Fe alone (Fig. 1C″), or in combination with 1 mM PQ and 15 mM Fe (Fig. 1C′″) in 1% GLU solution for different intervals of time. Red food dye (8 μl/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.
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 containing 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 saturated 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.
The movement deficits assay was performed on treated flies according to ref.  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] . Statistical analysis was performed on the PIs with the Student’s t test. Stalevo® (carbidopa, levodopa and entacapone), a drug amply medicated for the treatment of Parkinson’s disease, was used as a positive restorative drug against PQ toxicity.
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.
Polyphenols Protected and Maintained the Locomotion Function of Drosophila melanogaster Exposed to Paraquat
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, EC EGC, EGCG; 0.5 mM CA, and as a positive control, 0.5 mg/ml Stalevo® 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 effective 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, Stalevo® 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
Polyphenols Protected and Restored Locomotive Function in D. melanogaster Flies from Iron and Paraquat Exposure
It is known that the mechanism of paraquat toxicity in dopaminergic neurons is most likely mediated via oxidative stress . Indeed, paraquat (PQ2+) generates superoxide radicals (O2·−) by undergoing a NADH-dependent reduction to form a stable paraquat monocation (PQ+) radical that reacts very rapidly (k2 = 7.7 × 108 M s−1) with O2 to generate PQ2+ and O2·− radicals , which in turn produces H2O2 by either enzymatic or non-enzymatic dismutation reaction. Therefore, any molecule or compound capable of blocking either O2·− radicals or H2O2 generation might have potential antioxidant capacities . In this regard, polyphenols are the most abundant antioxidants in our diet . To establish conclusive evidence for the effectiveness of polyphenols in neurodegenerative 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  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 polyphenols (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 . 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 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 , 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 μM) 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 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 literature suggest that they may be pro-oxidant at certain concentrations [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 polyphenol supplementation, especially catechin derivatives EC, ECG, EGCG could increase survival time of D. melanogaster 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 , 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  are able to reduce the free radical DPPH· according to a DPPH· reduction test. Furthermore, phenolic acids have been shown to scavenge H2O2 . In accordance with these data, we have recently found that PG, EC and EGCG effectively attenuate mitochondrial damage against PQ-induced oxidative stress by scavenging O2·− radical and H2O2 thereby maintaining mitochondrial functionality by keeping high ΔΨm (M. Jimenez-Del-Rio, C. Velez-Pardo, J. Bustamante, S. Lores-Arnaiz, Unpublished 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, Stalevo® was included): FA=EGCG> EGC> PG=CouA= EC= Stalevo®> GA= CA at 12 h; GA> CA> EGCG= EC>EGC= Stalevo®> PG> FA= CouA at 24 h; and CouA> PG= FA=EGCG>CA= GA= EGC= Stalevo®> EC at 48 h. These data show that polyphenols positively affect 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 Stalevo®, a currently used medicine to treat patients with PD. Indeed, Stalevo® contains three active substances involved in the metabolism of dopamine: levodopa, which acts as a dopamine precursor; carbidopa, which is dopa decarboxylase inhibitor and entacapone, 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. melanogaster , we do not discard the possibility that the protective, rescue and locomotor activity of Stalevo® was associated, at least in part, to antioxidant activity of its components. However, the lack of polyphenols and Stalevo® bioavailability data in D. melanogaster discouraged us from concluding that a similar effect of movement amelioration in PD patients might be observed with polyphenol 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 polyphenol to iron is essential for antioxidant activity . On the other hand, increasing amounts of data suggest that iron might be involved in the pathogenesis of PD . Indeed, this metal has been found to accumulate in individual substancia nigra dopaminergic neurons from unfixed frozen post-mortem tissue of PD patients  and in substancia nigra from PD patients . However, it is yet unknown whether iron may be a causative or secondary factor in the disease process. Therefore, we initially investigated whether 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 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. melanogaster are highly homologous to those proteins involved in the metabolism of iron in humans . Second, dopaminergic 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 , rotenone  and iron [50–52]. One possible explanation 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 . However, further studies are needed to specifically evidence 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 . However, the phenolic acid PG and flavanols EGCG efficiently protected Drosophila exposed either to iron alone or in combination with iron and paraquat. 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 , but the capability of polyphenols to form iron-complexes and to scavenge free radicals might protect dopaminergic neurons from PQ-induced O2·− 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 regulators, 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 to suppress oxidative stress and to restore and/or maintain locomotor activity in PD patients.
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.