Effectiveness of γ-oryzanol in reducing neuromotor deficits, dopamine depletion and oxidative stress in a Drosophila melanogaster model of Parkinson's disease induced by rotenone

Accepted Manuscript Title: Effectiveness of ␥-oryzanol in reducing neuromotor deficits, dopamine depletion and oxidative stress in a Drosophila melanogaster model of Parkinson’s disease induced by rotenone Author: St´ıfani Machado Araujo Mariane Trindade de Paula Marcia R´osula Poetini Luana Meichtry Vandreza Cardoso Bortolotto Micheli Stefani Zarzecki Cristiano Ricardo Jesse Marina Prigol PII: DOI: Reference:

S0161-813X(15)00132-1 http://dx.doi.org/doi:10.1016/j.neuro.2015.09.003 NEUTOX 1862

To appear in:

NEUTOX

Received date: Revised date: Accepted date:

20-7-2015 4-9-2015 9-9-2015

Please cite this article as: Araujo SM, Paula MT, Poetini MR, Meichtry L, Bortolotto VC, Zarzecki MS, Jesse CR, Prigol M, Effectiveness of rmgamma-oryzanol in reducing neuromotor deficits, dopamine depletion and oxidative stress in a Drosophila melanogaster model of Parkinson’s disease induced by rotenone, Neurotoxicology (2015), http://dx.doi.org/10.1016/j.neuro.2015.09.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Effectiveness of γ-oryzanol in reducing neuromotor deficits, dopamine depletion

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and oxidative stress in a Drosophila melanogaster model of Parkinson’s disease

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induced by rotenone

4 Stífani Machado Araujo a, Mariane Trindade de Paulaa, Marcia Rósula Poetinia, Luana

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Meichtryb, Vandreza Cardoso Bortolotto b, Micheli Stefani Zarzeckia, Cristiano Ricardo

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Jesse a,b, Marina Prigol a,b*

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Moléculas Bioativas- LaftamBio Pampa- Universidade Federal do Pampa, Itaqui,

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CEP 97650-000,RS, Brazil.

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Laboratório de Avaliações Farmacológicas e Toxicológicas Aplicadas às

Department of Nutrition, UNIPAMPA- Campus Itaqui, RS, Brazil

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Toxicológicas Aplicadas às Moléculas Bioativas – LaftamBio Pampa. CEP 97.650-

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000 Itaqui, RS, Brazil. Tel.: +55-55-3433-1669.

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E-mail addresses: [email protected] (M.Prigol)

Corresponding

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Avaliações

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Abstract

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The γ-orizanol present in rice bran oil contains a mix of steryl triterpenyl esters of

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ferulic acid, which is believed to be linked to its antioxidant potential. In this study

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we investigated the neuroprotective actions of γ-orizanol (ORY) against the

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toxicity induced by rotenone (ROT) in Drosophila melanogaster. The flies (both

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genders) aged between 1 to 5 days old were divided into four groups of 50 flies

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each: (1) control, (2) ORY 25μM, (3) ROT 500μM, (4) ORY 25μM+ROT 500μM.

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Flies were concomitantly exposed to a diet containing ROT and ORY for 7 days

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according to their respective groups. Survival and behavior analyses were carried

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out in vivo, and ex vivo analyses involved acetylcholinesterase activity (AChE),

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determination of dopaminergic levels, cellular viability and mitochondrial

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viability, activities of superoxide dismutase (SOD), catalase (CAT), glutathione s-

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transferase (GST), reactive species levels (RS), lipid peroxidation (TBARS) and

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contents of total thiols and non- proteic thiols (NPSH). Our results show for the

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first time that ORY not only acts as an endogenous activator of the cellular

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antioxidant defenses, but it also ameliorates rotenone induced mortality, oxidative

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stress and mitochondrial dysfunction. Our salient findings regarded the restoration

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of cholinergic deficits, dopamine levels and improved motor function provided by

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ORY. These results demonstrate the neuroprotective potential of ORY and that this

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effect can be potentially due to its antioxidant action. In conclusion, the present

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results show that ORY is effective in reducing the ROT induced toxicity in D.

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melanogaster, which showed a neuroprotective action, possibly due to the presence

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of the antioxidant constituents such as the ferulic acid.

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Keywords: γ-oryzanol, rotenone, dopamine, Drosophila melanogaster, oxidative

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stress, neurotoxicty.

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1. Introduction The γ-oryzanol present in rice bran oil contains bioactive substances, a mix of steryl

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and other triterpenyl esters of ferulic acid. The microflora in the hind gut can hydrolyze

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these compounds, releasing ferulic acid for absorption or degradation (Zhao and

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Moghadasian, 2008). Some authors reported the beneficial effects of γ-oryzanol and

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their therapeutic applications, in vitro and in vivo models such as the inhibition of tumor

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growth (Pintha et al., 2014), reduction in plasma cholesterol levels (Wang et al., 2014),

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improvement of glucose intolerance (Kozuka et al., 2012), stimulation of

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osteoblastogenesis (Muhammad et al., 2013) and antioxidant properties (Juliano et al.,

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2005; Spiazzi et al., 2013; Wang et al., 2014). In the literature, there is also evidence of

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the neuroprotective effect of γ-oryzanol, related to one of its constituents, the ferulic

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acid. This polyphenol has protective function against neurodegenerative diseases such

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as Alzheimer’s disease, Parkinson's disease and stroke (Cheng et al., 2008).

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Late onset neurodegenerative diseases represent a major public health concern, since

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the mean age of world population is steadily increasing. Parkinson's disease (PD) is a

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common neurodegenerative syndrome characterized by degeneration of dopaminergic

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neurons in the substantia nigra (Feany et al., 2000). Moreover evidence suggests that α-

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synuclein inclusions and COX-2 up regulation contribute to mitochondrial dysfunction

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and neuroinflammation in PD. Increased expression of COX-2 has been reported to

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cause dopamine oxidation, which in turn, triggers oxidative stress and α-synuclein

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accumulation (Gupta and Benzeroual, 2013). Oxidative stress has been assigned as an

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important factor in the progression of PD (Muñoz-Soriano and Paricio, 2011).

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Increasing evidence suggests that PD may be associated with mitochondrial dysfunction

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through a variety of routes, including the generation of free radicals, inflammation and

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dysfunction of complex I of the mitochondrial respiratory chain (Greenamyre et al.,

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2001; Winklhofer et al., 2010). However, most cases are sporadic and of unknown

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etiology, or they are thought to be a result of exposure to environmental toxins such as

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toxic agents i.e. rotenone, paraquat, among others. Knowing that the oxidative stress

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and dopaminergic loss plays a central role in PD disease, several studies have been

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conducted to investigate the potential neuroprotective effect of antioxidants in models

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of PD, mainly medicinal plants, or the use of bioactive plant components, since they are

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normally free of adverse effects.

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Rotenone, a well-established mitochondrial toxin is often used as a chemical model,

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since it reproduces some important aspects of PD pathology both in Drosophila and

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rodent models (Canon and Greenamyere, 2010; Tamilselvam et al., 2013; Manjunath et

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al., 2015). The rotenone is a potentially toxic agent, which belongs to the class of

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rotenoids (natural cytotoxic compounds extracted from tropical plants such as Derris

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elliptica) and it is used as a pesticide (Tanner et al., 2011). By presenting lipophilic

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activity, it quickly reaches cell organelles like mitochondria, which acts as a specific

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inhibitor, of high affinity of the mitochondrial NADH dehydrogenase (complex I)

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(Cicchetti et al., 2009). According to Perfeito and Rego (2012), rotenone is considered

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to be responsible for dopaminergic cell death, and for causing effects such as increased

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production of free radicals and consequent oxidative stress-mediated increase in the

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mitochondria.

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Invertebrate animals such as Drosophila melanogaster have been an efficient study

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model and it has been widely explored as a powerful genetic tool for understanding

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complex biological problems. Many basic biological, physiological, and neurological

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properties are conserved between mammals and Drosophila melanogaster, and nearly

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75% of human disease-causing genes are believed to have a functional homolog in the

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fly (Pandey and Nichols, 2011). Recently, many researchers have used these flies as a

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model to understand various neurodegenerative diseases, since they have homology

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between five of the six genes related to PD in humans (Whitworth et al., 2006; Benton

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et al., 2008; Bagatini et al., 2011). This study aimed to investigate the possible

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neuroprotective effect of γ-oryzanol on behavioral and biochemical changes caused by

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chronic exposure of Drosophila melanogaster to rotenone.

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2. Materials and Methods

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2.1. Materials and Fly Culture Condition

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γ-oryzanol (ORY) was purchased from Tokyo Chemical Industry Co., Ltd.

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(Tokyo, Japan). Rotenone was purchased from Sigma-Aldrich (St. Louis, MO, USA).

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All other reagents were of analytical grade from the Campus of UNIPAMPA.

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Drosophila melanogaster (D. melanogaster) wild-type (strain Harwich) was

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obtained from the National Center species, Bowling Green, Ohio, USA. The hatched

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flies were maintained for about 3 days in an incubator with controlled temperature of 25 4 Page 4 of 29

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° C and 30-50% humidity under a light / dark cycle of 12 h fed on standard medium

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(1% cornflour w/v; 1% yeast w/v beer, 1% w/v wheat germ 2 % w / v sucrose, 1% w/v

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milk powder; 0.08% w/v nipagin).

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2.2. Experimental Protocol 2.2.1. Rotenone exposure and γ-oryzanol treatment

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D. melanogaster (both genders) of 1 to 5 days old were divided into four groups

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of 50 flies each: (1) control, (2) ORY 25μM, (3) ROT 500 μM, (4) ORY 25μM +ROT

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500 μM. Flies were concomitantly exposed to a diet containing ROT and ORY for 7

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days according to their respective groups.

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ROT (500 μM) was diluent in ethanol and it concentration was similar to used

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by Hosamani et al., (2009) and Sudati et al., (2013). ORY (25μM) was diluent in

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sucrose. ORY concentration was chosen based in a pilot experiment where flies were

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exposed to ORY at various concentrations such as of 25 µM, 50 µM and 75 µM to

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determine the effect of ORY alone on the survival of flies during the experimental

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period. However, only one of the concentrations was considered satisfactory when

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assessing mortality and the behavioral test for negative geotaxia of flies, thus, exposure

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to the concentration used was 25 µM.

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ROT and ORY were added into the food’s flies at final concentration of 500 μM

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and 25 μM, respectively. The total food medium contained a volume of 1% of ethanol

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and 1% sucrose, ORY, ROT or ORY plus ROT. Two controls were used (with and

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without 1% of ethanol and 1% sucrose). Only the control with 1% of ethanol and 1%

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sucrose is showed in the results because there was no statistical difference between

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these groups in all parameters evaluated. The diet during the treatment consisted in 1%

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yeast w/v beer, 2 % w/v sucrose, 1% w/v milk powder, 1% agar w/v; 0.08% w/v

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nipagin.

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2.3. In vivo assays

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2.3.1. Survival rate

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The survival rate was evaluated by counting daily the number of living flies until

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the end of the experimental period (7 days). Around 150 flies per group were included

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in the survival data and the total number of flies represents the sum of three independent

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experiments (50 flies/each treatment repetition). 5 Page 5 of 29

2.3.2. Negative geotaxis

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Negative geotaxis of the flies was determined according to Jimenez del-rio et al.,

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(2010) by recording the time spent by each fly to achieve a height of 8 cm measured

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from the bottom of a glass test tube with a diameter of 1.5 cm. The test is repeated five

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times for each fly, and 15 flies are separated from each group to be evaluated, and the

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data were analyzed according to the averaging time. The total number of flies (60 per

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group) represents the sum four independent experiments.

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2.3.3. Negative geotaxis Test Base Top

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Locomotor activity of flies was determined based on negative geotaxis behavior

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assay as previously described by Coulom and Birman (2004) with some modifications.

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The flies were exposed to ROT as described above. Following exposure, 10 flies (both

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genders) were classified and received brief anesthesia with ice and were then transferred

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to a Falcon tube (15 cm length and 1,5 cm in diameter). After 30min of recovery from

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ice, flies were gently tapped to the bottom of the tube, and the number of flies that

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climbed up to the 6 cm mark of the column (i.e., the top) in 5 sec as well as those that

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remained below the mark, were counted separately. The procedure was repeated five

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times per group at 1min intervals. The total number of flies (40 per group) represents

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the sum four independent experiments.

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2.3.4. Test open field

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To evaluate the behavior and exploratory activity of the fly, 15 flies were used in

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each group and these were kept in a petri dish divided by a square centimeter, which can

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be covered by a petri dish, described by (Hirth 2010). Fly activity and movement were

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recorded and evaluated and the resulting trajectory for the time (60 s) was calculated

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according to the number of squares crossed/explored by each fly analyzed in each

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group. The total number of flies (60 per group) represents the sum four independent

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experiments.

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2.4. Ex vivo assays 2.4.1. Homogenized preparation

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About 20 flies per group were immobilized by freezing in ice for approximately

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1 min, then they were manually homogenized in ice-cold HEPES buffer (20 mM, pH

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7.0), 10: 1 (flies / volume (µl)) with or without head according to each analysis to be

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performed after centrifuged rotation according to each analysis to be performed, and the

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supernatant was removed and used for biochemical assays. All experiments were

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performed in triplicate.

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2.4.2. Analysis of dopamine concentrations by HPLC

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Twenty flies of each group had their heads separated from their bodies using a

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sharp blade. The head was homogenized in 100 µl of sodium phosphate buffer (0.1M,

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pH 7.4) containing 1mM EDTA, followed by centrifugation at 25009g for 10 min at 4 °

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C. The supernatant was directly used to estimate the levels of dopamine by HPLC

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(Dalpiaz et al., 2007).

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2.4.3. Evaluation of cell viability

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Cellular viability was measured by two different methods. Firstly, cellular

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viability was measured using. Cellular viability was measured using 3-(4,5-

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dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay as

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described by (Hosamani and Muralidhara, 2013). The flies were incubated in MTT for

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60 min (37 °C), after the MTT removal, the sample was incubated in DMSO for 30 min

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(37 °C). The absorbance from formazan dissolution by the addition of DMSO was

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monitored in an EnsPireR multimode plate reader (Perkin Elmer, USA) at 540 nm.

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The second method used was the resazurin reduction assay. The method is based

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in the ability of viable cells to reduce resazurin to resorufin, a fluorescent molecule.

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(Franco et al., 2009). Groups of 20 flies were homogenized in 1 ml 20 mM Tris buffer

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(pH 7.0) and centrifuged at 3570 rpm for 10 min at 4 ° C. After that, the supernatant

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was incubated in ELISA plates with 20 mM buffer Tris (pH 7.0) and resazurin for two

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hours. Fluorescence was recorded using EnsPireR multimode plate reader (Perkin

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Elmer, USA) at ex 579nm to em584nm.

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2.4.4. Evaluation of Mitochondrial viability

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Mitochondrias were isolated from the whole body of flies by modified

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differential centrifugation method (Hosamani and Muralidhara, 2013). Briefly, flies

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were homogenized in ice-cold Tris-sucrose buffer (0.25 M, pH 7.4) (60 mg fly tissue

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homogenized in 1000 μl buffer) and centrifuged at 1000 x g for five minutes (4° C).

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Mitochondria were obtained by centrifuging the postnuclear supernatant at 10000 × g

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for 10 minutes (4° C). The pellet was washed in mannitol–sucrose–HEPES buffer and

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resuspended in 200 μl of suspension buffer.

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Mitochondrial fraction (200 microlitros) were with 3-[4,5-dimethylthiazol-2-yl]-

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2,5-diphenyltetrazolium bromide) MTT (x% solution) for 30 minutes at 37° C. After

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that, the samples were centrifuged at 10000 x g for five minutes. The pellet was

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dissolved in Dimethyl sulfoxide (DMSO), incubated for 30 minutes at 37° C and the

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absorbance was measured at 540 nm. Results were expressed as percentage of the

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control.

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2.4.5. Thiol Determination

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Thiol Protein and non-protein were estimated based on spectrophotometry using

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the Ellmann (1959) reagent. Ten flies in each group were manually homogenized after

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the homogenate was removed and added to 0.5M PCA and centrifuged at 10.000 rpm

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for 5 min at 4 ° C.

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For non-protein thiol measures, the supernatant was used (the pellet should be

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reserved for later use) and added 5,5’ dithiobis-2-nitrobenzoic acid (DTNB) 5mM

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awaited 15 min at room temperature protected from light and the reading was doneby

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spectrophotometer at 412 nm. For non-protein thiol measures of previous samples the

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pellet was resuspended in Tris/HCl 0.5M pH 8.0 buffer, the supernatant was removed

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and added to 5 mM DTNB, awaited15 minutes at room temperature protected from light

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and the reading was done by the spectrophotometry at 412 nm.

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2.4.6. Determination of Reactive species levels (RS) and Lipid Peroxidation

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For quantification of reactive species generation, a total of 20 flies were

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anesthetized on ice and homogenized in 1 ml 10 mM Tris buffer, pH 7.4. The

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homogenate was centrifuged at 1000 x g for 5 minutes at 4 ° C and the supernatant was

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removed for assay quantification of 2 ', 7'-dichloroflurescein diacetate (DCF-DA)

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oxidation, as a general index of oxidative stress according to the protocol of Pérez8 Page 8 of 29

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Severiano et al., (2004). The fluorescence emission of DCF resulting from DCF-DA

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oxidation was monitored after one hour at

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multimode plate reader (Perkin Elmer, USA). Results represent the mean of three

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independent experiments. In each experiment, each treatment was done in duplicate.

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Lipid peroxidation was measured by TBARS. Ten flies in each group were

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homogenized and centrifuged at 1000 rpm for 10 min at 4 ° C. The supernatant was

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removed and added to TBA (0.8% pH 3.2), acetic acid (20%, pH 3.5), sodium lauryl

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sulfate (SDS) (8%). The mixture was incubated for two hours at 95 ° C. Immediately

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after the absorbance, it was measured at 532 nm (Ohkawa et al., 1979).

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2.4.7.1.

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2.4.7. Activities of Antioxidant Enzymes Determination of catalase activity

The catalase activity was measured following the method of (Aebi, 1984). Ten

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flies in each group were homogenized and centrifuged at 14000 rpm for 30 min at 4 ° C.

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Containing solution was prepared (0.25M KPi buffer / 2.5 mM EDTA pH 7.0, 30%

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H2O2, and water triton X-100), This solution was added to the supernatant and the

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sample readings were taken at 240 nm for 1 min .

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2.4.7.2.

Determination of superoxide dismutase activity

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SOD activity was measured by monitoring the inhibition of quercetin auto

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oxidation. Ten flies in each group were homogenized and centrifuged at 14000 rpm for

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30 min at 4 ° C. A solution containing was prepared containing (0.25M KPi buffer / 0.1

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mM EDTA pH 10.0 and N, N, N, N-tetramethyl ethylenediamine (TEMED) at the time

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of the reading the supernatant of the sample with quercetin was added to the solution

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and this Reading was performed for 2 min at 406 nm. Expressed in terms of amount of

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protein required to inhibit 50 % of quercetin auto oxidation (Kostyuk and Potapovich

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1989).

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2.4.7.3.

Determination of Glutathione-S-transferase activity (GST)

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Assay of GST activity was performed according to the procedure of (Habig et

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al., 1974) using 1-chloro-2–4-dinitrobenzene (CDNB). Ten flies in each group were

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homogenized and centrifuged at 14000 rpm for 30 min at 4 ° C. Containing solution

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was prepared (0.25M Kpi buffer / EDTA (2.5 mM, pH 7.0), distilled water and 100mM

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GSH) in the sample and the supernatant was added 50 mM CDNB and its reading was

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taken at 340 nm for 2 min. 9 Page 9 of 29

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2.4.7.4.

Activity of acetylcholinesterase (AChE)

Acetylcholinesterase activity was determined according to the method of

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Ellmann (1961). Ten flies in each group were homogenized and centrifuged at 1000 rpm

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for 5 min at 4 ° C. The same reaction was prepared containing (0.25M KPi buffer, pH

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8.0 and 5.5 dithiobis-2-nitrobenzoic acid (DTNB 5mM)) is added to the supernatant

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solution sample and to acetylthiocholine 7,25mM (2, 1mg / ml), and the reading was

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done for 2 min at 412nm. The enzymatic activities were expressed as nmoles of

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hydrolyzed substrate / min /mg protein.

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2.4.8. Protein determination

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Protein concentration was measured by the method of Bradford (1976) using

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bovine serum albumin as standard. 2.5. Statistical analysis

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Lifespan measurement was determined by comparing the survival curves with a

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log-rank (Mantel-Cox) test. Other statistical analysis was performed using two-way

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ANOVA followed by Newman-Keuls post hoc test where appropriate. Differences were

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considered significant between groups at p <0.05 using the GraphPad Prism5 program.

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3. Results 3.1. ORY improves the survival rate of D. melanogaster Exposure of adult flies to ROT resulted in lethality over a 7 day experimental

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period (Fig. 1) when compared to the control group. The lifetime of flies receiving

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ORY was up to 7 days. However, in the ROT group this survival rate drops to 3 days.

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When compared with the ROT group, the mortality rate was lower for the flies treated

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simultaneously with ORY, indicating its ability to protect against ROT induced

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mortality. (p <0.05).

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Fig. 1. Effect of γ-oryzanol (ORY) on survival rate of flies exposed to rotenone (ROT). Data were

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collected every 24 h for each group during 7 days. The total number of flies (150 per group) represents

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the sum of three independent experiments. Lifespan measurement was determined by comparing the survival curves Mantel-Cox log-rank test and multiple comparisons were corrected using the Bonferroni method.

3.2. Locomotor performance and Acetylcholinesterase activity

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Exposure of adult flies to ROT caused impact deleterious on locomotor behavior

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as reductions in the climbing rate compared with the control. This effect was abolished

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by the treatment with ORY, once flies from this group had better performance climbing

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(Fig. 2A and 2B). Regarding the open field test, the group exposed to ROT showed less

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exploratory activity when compared to the control group (Fig. 2C, p< 0.05) indicating

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locomotor deficit. Acetylcholine is the primary excitatory neurotransmitter in the central

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nervous system, in parallel with the behavioral parameter; its activity was also assessed

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(Kim and Lee, 2013). As shown in Fig.3, there was a significant inhibition of AChE 11 Page 11 of 29

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activity (p <0.05) in flies exposed to ROT, compared to the control group. This ROT

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effect was annulled by the treatment with ORY.

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A)

B)

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C)

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Fig. 2. Effect of γ-oryzanol (ORY) on geotaxic response (climbing) and exploratory activities of flies

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exposed to rotenone (ROT) during 7 days. Graphic (A) negative geotaxis assay, Graphic (B) negative

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geotaxis test base top and Graphic (C) open-field. 15 flies per group were included for the

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negative geotaxis and open-field tests (total of 60 flies) and 10 flies per group were

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included for the negative geotaxis test base top (total of 40 flies). These values represent the

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sum of four independent experiments. Values are mean ± SE. Significance determined by two-

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way analysis of variance (ANOVA) followed by Newman-Keuls test. *Significant difference in relation to the control group; #Significant difference between ROT and ROT+ ORY (p < 0.05).

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Fig.3. Effect of γ-oryzanol (ORY) on rotenone (ROT) induced alterations in activity of

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acetylcholinesterase of adult Drosophila melanogaster. Values are mean ± SE (n = 20 flies per replicate,

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3.3. Effect of ORY on the depletion of the levels of dopamine (DA) induced

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three replicates used). Significance determined by two-way analysis of variance (ANOVA) followed by Newman-Keuls test. *Significant difference in relation to the control group; #Significant difference

ROT

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between ROT and ROT+ ORY (p < 0.05).

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The flies of the ROT group showed a depletion of 42% in the levels of dopamine

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in the head compared to the control group. Treatment with ORY protected against the

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reduction of dopamine levels in the head caused by ROT in flies. (Fig.4 p <0.05).

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Fig.4. Effect of γ-oryzanol (ORY) on rotenone (ROT) induced alterations in activity of dopamine

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levels in the head of adult Drosophila melanogaster. Values are mean ± SE (n = 20 flies per replicate, three replicates used). Significance determined by two-way analysis of variance (ANOVA) followed by Newman-Keuls test. *Significant difference in relation to the control group; #Significant difference between ROT and ROT+ ORY (p < 0.05).

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3.4. Effect of ORY on Cell viability by MTT reduction in homogenate of flies exposed to rotenone

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Exposure to ROT caused a significant decrease on MTT reduction in the fly

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homogenates, confirming the reduction in cell viability. This effect was abolished by

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the ORY treatment (Fig. 5A p<0,05).

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exposed to rotenone

Exposure to ROT caused a significant decrease on MTT reduction on

cr

372

3.5. Effect of ORY on Mitochondrial Viability by MTT reduction in flies

mitochondria. This effect was abolished by the ORY treatment (Fig. 5B p<0,05). A)

B)

an

376

us

371

377

M

378

ed

379 380

ce pt

381 382

Fig.5. Effect of γ-oryzanol (ORY) exposure on cell viability by MTT of flies exposed to rotenone

383 384 385 386 387

(ROT) during 7 days. Graphic (A) cell viability in homogenate of flies and Graphic (B) cell viability

388

3.6. Effect of ORY on Resazurin Reduction Assay in homogenate of flies

389

mitochondrial. Values are mean ± SE (n = 20 flies per replicate, three replicates used). Significance determined by two-way analysis of variance (ANOVA) followed by Newman-Keuls test. *Significant

Ac

difference in relation to control group, #Significant difference between Rot and Rot + ORY groups. Results were expressed as the percentage (%) of the control group (mean ± standard deviation p < 0.05).

exposed to rotenone

390

Cell viability measured by the rezaruzin reduction test showed a significant drop

391

in cell viability for the group exposed to ROT compared to the control group,

392

confirming the toxicity of ROT cell level. This effect can be reversed by a treatment

393

with ORY (Fig.6, p <0.05).

14 Page 14 of 29

ip t cr

394

Fig.6. Effect of γ-oryzanol (ORY) exposure on cell viability in homogenate of flies treated with rotenone

401

3.3 Oxidative stress and Antioxidants Defenses

us

395 396 397 398 399 400

(ROT) performed by rezaruzin reduction assay. Values are mean ± SE (n = 20 flies per replicate, three replicates used). Significance determined by two-way analysis of variance (ANOVA) followed by

an

Newman-Keuls test. *Significant difference in relation to control group, #Significant difference between Rot and Rot + ORY groups. Results were expressed as the percentage (%) of the control group (mean ±

M

standard deviation p < 0.05).

Oxidative stress has been assigned as an important factor in the progression of

403

PD. Here we quantify DCFDA oxidation as a general indicator of oxidative stress and

404

TBARS as an indicator of lipid peroxidation (Fig. 8A and 8B). Flies treated with ROT

405

had a significant increase in the production of reactive species measured by the

406

oxidation of DCFDA compared to the control group. Also, there was an increase in lipid

407

peroxidation (LPO) and its product malondialdehyde (MDA) that can be used as an

408

indicator of the action of free radicals in the body, in the group receiving ROT

409

compared to the control group. Co-exposure to ORY and ROT was able to reduce MDA

410

levels and DCFDA oxidation induced by the rotenone.

ce pt

Ac

411

ed

402

The ROT group had an inhibition on SOD, CAT and GST activity when

412

compared to the control group. Treatment with ORY was effective to reduce the

413

inhibition of catalase (CAT), superoxide dismutase (SOD) and glutatione S-transferase

414

(GST) activities to control levels (Fig. 7A, 7B and 7C).

415 416

The content of protein thiols and nonprotein thiols were not altered in all groups tested (Fig. 9A and 9B).

15 Page 15 of 29

B)

us

cr

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A)

ce pt

ed

M

an

C)

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417

16 Page 16 of 29

418

Fig. 7. Effect of γ-oryzanol (ORY) for 7 days on alterations induced by rotenone (ROT) the

419 420 421 422 423 424

activities of antioxidant enzymes in the whole homogenate body of adult Drosophila melanogaster. Graphic (A) superoxide dismutase (SOD) ,Graphic (B)

catalase (CAT) , and Graphic (C)

glutathione-S-transferase activity (GST). Values are mean ± SE (n = 20 flies per replicate, three replicates used). Significance determined by two-way analysis of variance (ANOVA) followed by Newman-Keuls test. *Significant difference in relation to the control group; #Significant difference

ip t

between ROT and ROT+ ORY (p < 0.05).

425 A)

B)

cr

426 427

us

428

an

429 430

M

431 432

440 441

Fig.8. Effect of γ-oryzanol (ORY) for 7 days on alterations induced by rotenone (ROT) in markers of oxidative stress. Graphic (A) reactive species levels (RS) and (B) levels of lipid

ce pt

endogenous

peroxidation (LPO) . Values are mean ± SE (n = 20 flies per replicate, three replicates used). Significance determined by two-way analysis of variance (ANOVA) followed by Newman-Keuls test. *Significant difference in relation to the control group; #Significant difference between ROT and ROT+ ORY (p < 0.05).

Ac

434 435 436 437 438 439

ed

433

A)

B)

442 443 444 445

17 Page 17 of 29

446 447 448 449 450 451

Fig. 9. Effect of γ-oryzanol (ORY) on protein thiols graphic (A) and non-protein thiol graphic (B)

452

4. Discussion

content in homogenate of flies exposed to rotenone (ROT). Results were expressed as the percentage (%) of the control group (mean ± standard deviation p < 0.05). Values are mean ± SE (n = 20 flies per replicate, three replicates used).Significance determined by two-way analysis of variance (ANOVA) followed by Newman-Keuls test. *Significant difference in relation to the control group; #Significant

ip t

difference between ROT and ROT+ ORY (p < 0.05).

Our results demonstrate for the first time the protective effect of ORY in a

454

model of Parkinson's disease induced by the exposure to rotenone in D. melanogaster.

455

Importantly, the ORY acted on this model by restoring dopamine levels in heads of flies

456

and the activity of the enzyme acetylcholinesterase and improving motor function of

457

flies. Furthermore, the compound improved antioxidant defenses, preventing oxidative

458

stress, mitochondrial dysfunction and preventing the lethality induced by rotenone. In

459

this way, in the literature we find studies that indicate a neuroprotective role of

460

constituents of ORY which consists of a mixture of ferulic acid esters, which give the

461

ORY its antioxidant potential (Paucar-Menacho et al., 2007; Sultana, 2012; Maruf et al.,

462

2015). Ferulic acid acts as an antioxidant agent against radical oxidation in neuronal cell

463

culture systems (Kanski et al., 2002), it also exerts a neuroprotective effect through the

464

inhibition of apoptosis in focal cerebral ischemic injury (Koh,2015).

465

administration of ferulic acid for 4 weeks protects mice against learning and memory

466

deficits induced by centrally administered β-amyloid1-42 (Kim et al., 2004), it also

467

decreases the expression of active caspase-3 in the rat striatum (Cheng et al., 2008) and

468

prevented increases in interleukin-1a immunoreactivity and in the levels of endothelial

469

nitric oxide synthase and 3-nitrotyrosine in the activated astrocytes of the mouse

470

hippocampus (Cho et al., 2005). Besides that, it exhibits neuroprotection against striatal

471

neuronal cells subjected to oxidized low-density lipoproteins (Schroeter et al., 2000).

us

an

M

ed

ce pt

Long-term

Ac

472

cr

453

According to our results the ORY was effective in reducing neuromotor deficits,

473

geotaxia negative tests (climbing), and open field test (rating exploratory capacity).Flies

474

with locomotor deficits tend to stay in the bottom of the glass column and they have the

475

ability to coordinate their legs in a normal manner. This phenotypic expression is

476

explained by the requirement for high power of the muscles that are rich in

477

mitochondria (Hosamani et al., 2009). Although speculative, it is likely that

478

mitochondrial machinery may have been disengaged due to severe inhibition of 18 Page 18 of 29

complex I caused by rotenone toxicity (Cicchetti et al., 2009). In parallel with the

480

behavioral parameter, the activity of acetylcholinesterase (AChE) is well described

481

being used in the verification of the efficacy of treatment for PD and other

482

neurodegenerative diseases (Hosamani et al., 2010) this is an enzyme that participates in

483

cholinergic neurotransmission and it is able to break down. The neurotransmitter

484

acetylcholine ends this process, when this activity is inhibited it leads to the

485

accumulation of the neurotransmitter at the synapses (Cálic et al., 2006; Pohanka et al.,

486

2011). AChE activity is inhibited by several compounds among them, flies exposed to

487

the ROT had decreased AChE activity in our study, but the treatment with the ORY was

488

able to prevent this inhibition. As reported by Szwajgier and Borowiec (2012) in a study

489

with mices, ferulic acid prevented a decrease in acetylcholine levels in the cortex caused

490

by intracerebroventricular injection of β-amyloid and protected mice against cognitive

491

impairments, as demonstrated in the passive avoidance, Y-maze (spatial memory) and

492

water maze tests.

an

us

cr

ip t

479

In the central nervous system, it is was possible to identify six dopaminergic

494

neuronal sets (PPM3, PPL2, PPL1, PAM, and PAL, PPM 1/2) that are normally present

495

in each hemisphere of the brain of Drosophila adult (Coulom and Birman, 2004).

496

Drosophila melanogaster also contain dopamine transporter (dDAT) (Ueno et al., 2014)

497

and vesicular monoamine transporter (VMAT) gene ( Greer et al., 2005), responsible

498

for dopamine homeostasis. The dopamine is involved in the control of locomotion,

499

cognition, affect and neuroendocrine secretion (Giros et al., 1996). In Parkinson's

500

disease, the accompanying dopaminergic depletion is traditionally considered one of the

501

underlying mechanisms that contribute to the cardinal motor symptoms of bradykinesia,

502

rigidity and tremor (Chong et al., 2015). Neurotoxins such as rotenone can lead to

503

similar signs and symptoms of PD as this disorder is associated with mitochondrial

504

dysfunction due to defects in complex I of the electron transport chain (Cicchetti et al.,

505

2009). According to Hastings (1995) and Jahromi et al., (2015) complex I inhibition

506

decreased antioxidant levels, and increased iron levels, dopaminergic neurons may be

507

under a greater amount of oxidative stress than other neurons in the brain, because they

508

contain dopamine. The monoamine oxidase metabolism produces H2O2 that can

509

participate in Fenton type reaction with Fe (II) to generate ROS involved in neuronal

510

death in the substantia nigra.

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19 Page 19 of 29

Dopamine metabolism is a complex biochemical system, which includes the

512

synthesis, storage, release, reuptake and degradation of neurotransmitter (Qi et al.,

513

2008). In our study, the group exposed to ROT had a significant depletion of dopamine

514

levels in the head of flies. Previous studies have reported several mechanisms that

515

justify a decrease in dopamine levels in the head flies treated with rotenone, among

516

which are loss of dopaminergic neurons in brain (Coulom and Birman, 2004) and

517

decrease in vesicular monoamine transporter (VMAT) (Lawal et al., 2010).

518

Surprisingly, ORY was capable to prevent dopamine depletion, proving to be a potential

519

neuroprotective agent, which we believe to have a significant correlation between

520

locomotor dysfunction and deficiency of dopamine because the dopamine neurons of

521

the brain comprise the black substance release DA and facilitate movement (Hauser and

522

Hastings, 2013). According to Gupta and Benzeroual (2013), ferulic acid has the ability

523

to inhibit COX-2 enzyme, a mechanism that prevents the oxidation of dopamine, and α-

524

synuclein accumulation giving the ferulic acid component of ORY a neuroprotective

525

effect.

M

an

us

cr

ip t

511

Oxidative stress is considered an important factor for neurodegeneration in PD

527

(Muñoz-Soriano and Paricio, 2011). In the present study, we found significant induction

528

of oxidative stress in flies exposed to rotenone as evidenced by the sharp rise in the

529

levels of MDA and ROS along with significant increases in antioxidant enzyme

530

activities such as CAT, SOD and GST. Taken together, the results suggest the

531

involvement of oxidative stress-based model of rotenone with D. melanogaster.

532

Depletion of these cellular levels of antioxidants was reduced with treatment with ORY,

533

resulting in complete reduction of oxidative markers which clearly indicates the

534

antioxidant capacity of ORY. As also described by (Sultana, 2012), ferulic acid has free

535

radical scavenging activity towards hydroxyl radical, peroxynitrite, superoxide radical

536

and oxidized low-density lipoprotein, the hydroxyl group in the ferulic acid can readily

537

form a resonance stabilized phenoxy radical and this is a key to its antioxidant property

538

(Castelluccio et al.,1996; Ogiwara et al., 2002). Ferulic acid can also protect biological

539

membranes from lipid peroxidation and neutralized peroxyl and alkoxyl radicals

540

(Trombino et al., 2004). The contents of NPSH and PSH did not change and it is

541

consistent with previous observations (Hosamani et al., 2010; Manjunath et al., 2015).

Ac

ce pt

ed

526

542

In the current study, the flies exposed to ROT showed decreased cell viability

543

using two different methodologies, indicating that the viability of the cells was 20 Page 20 of 29

compromised, resulting in high mortality of the flies during the exposure period of 7

545

days. The treatment with ORY resulted in higher cell viability, low incidence of

546

mortality of flies treated with ORY, which clearly indicates the presence of protective

547

bioactive compounds in ORY which may be responsible for the suppression of free

548

radicals or an increase in the regulation of antioxidant defenses. The literature reported

549

that the ferulic acid effect against apoptosis may be the overall effect of both scavenging

550

of reactive oxygen species directly and chelation of iron to inhibit the Fenton reaction

551

indirectly, ferulic acid can chelate the ferrous ion and decrease the formation of

552

hydroxyl radicals through the inhibition of the iron dependent Fenton reaction (Zhang,

553

2003).

cr

ip t

544

In this study, we first assessed the effects of ORY in a model of

555

neurodegenerative diseases such as the Parkinson's disease ROT induced toxicity in D.

556

melanogaster. Our data demonstrate the effectiveness of ORY in reducing the

557

deleterious effects of ROT toxicity in both behavior parameters, dopamine levels and

558

oxidative stress after in vivo exposure of D. melanogaster, these properties provide a

559

mechanism for preventing the toxicity caused by ROT. In conclusion, the present results

560

show that ORY is effective in reducing the toxicity induced by rotenone in D.

561

melanogaster, showing neuroprotective action, possibly due to the presence of the

562

antioxidant constituents such as ferulic acid.

563

ACKNOWLEDGMENTS

564

The financial support by FAPERGS, CAPES and CNPq is gratefully acknowledged.

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Highlights

Protective effect of ɣ-oryzanol in a model of Parkinson's disease. ɣ-oryzanol restores dopamine levels in heads of Drosophila melanogaster. ɣ-oryzanol improved antioxidant defenses, preventing oxidative stress. ɣ-oryzanol demonstrates neuroprotective effect due to its antioxidant constituents.

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