Free Radical Research

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Exercise associated with γ-oryzanol

supplementation suppresses oxidative stress and
prevents changes in locomotion in Drosophila
melanogaster

Mustafa Munir Mustafa Dahleh, Stífani Machado Araujo, Vandreza Cardoso

Bortolotto, Franciane Cabral Pinheiro, Márcia Rósula Poetini, Elize Aparecida
Santos Musachio, Luana Barreto Meichtry, Shanda de Freitas Couto &
Marina Prigol

To cite this article: Mustafa Munir Mustafa Dahleh, Stífani Machado Araujo, Vandreza Cardoso
Bortolotto, Franciane Cabral Pinheiro, Márcia Rósula Poetini, Elize Aparecida Santos Musachio,
Luana Barreto Meichtry, Shanda de Freitas Couto & Marina Prigol (2021): Exercise associated with
γ-oryzanol supplementation suppresses oxidative stress and prevents changes in locomotion in
Drosophila�melanogaster, Free Radical Research, DOI: 10.1080/10715762.2021.1895992

To link to this article: https://doi.org/10.1080/10715762.2021.1895992

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FREE RADICAL RESEARCH
https://doi.org/10.1080/10715762.2021.1895992

ORIGINAL ARTICLE

Exercise associated with c-oryzanol supplementation suppresses oxidative

stress and prevents changes in locomotion in Drosophila melanogaster
Mustafa Munir Mustafa Dahleh , St
ıfani Machado Araujo , Vandreza Cardoso Bortolotto ,
Franciane Cabral Pinheiro , M
arcia Ro

sula Poetini , Elize Aparecida Santos Musachio ,
Luana Barreto Meichtry , Shanda de Freitas Couto and Marina Prigol
Laboratory of Pharmacological and Toxicological Evaluations Applied to Bioactive Molecules, LaftamBio - Federal University of Pampa,
Itaqui, Brazil

ABSTRACT ARTICLE HISTORY

Association to early mortality and sedentarism was already demonstrated in the literature; never- Received 11 November 2020
theless, some possible biochemical mechanisms around physical inactivity still need answers. The Revised 20 January 2021
use of an invertebrate model, such as Drosophila melanogaster, can reproduce reliable responses Accepted 23 February 2021
in inducing an exercise protocol with exogenous antioxidant supplementation. This study main
KEYWORDS
evaluates the effect of exercise (EXE) associated with c-oryzanol (ORY) supplementation to Exercise; locomotor
improve locomotor behavior, antioxidant defenses, and survival in Drosophila melanogaster. Two- behavior; Drosophila
day old flies were submitted to a protocol for seven days, divided into five groups: Control, melanogaster; antioxidant;
Movement-Limited Flies (MLF), EXE, ORY [25 mM], and EXE þ ORY [25 mM]. The survival rate was biochemical parameters
evaluated, followed by open field and negative geotaxis. Flies were euthanized and subjected to
analysis for acetylcholinesterase (AChE) and antioxidant enzymes activity, glycidic and lipid
parameters, body weight, reactive species (RS), and lipid peroxidation. EXE and EXE þ ORY flies
showed increased survival and locomotor activity, improved glycidic and lipid parameters, with a
lower RS production, and increased antioxidant defenses compared to Control, and EXE þ ORY
when compared to the EXE group, obtained an increase in the ratio of protein levels/body
weight, decreased ratio of triglyceride levels/body weight and decreased lipid peroxidation.
However, MLF showed less survival and decreased locomotor activity, possibly due to increased
AChE activity and reduced antioxidant defenses. The EXE and EXE þ ORY demonstrate effective
results in maintaining endogenous defenses, with increased locomotor activity, supporting evi-
dence on EXE benefits, and supplementation with antioxidant compounds face of
health paradigms.
HIGHLIGHTS
 New protocol system of exercise on Drosophila melanogaster model.
 ORY demonstrates synergistic effect with EXE.
 Exercise with ORY supplementation increases locomotor behavior.
 Exercise with ORY supplementation decrease oxidative damages on flies.
GRAPHICAL ABSTRACT

CONTACT Marina Prigol marinaprigol@unipampa.edu.br Laboratory of Pharmacological and Toxicological Evaluations Applied to Bioactive
Molecules, LaftamBio – Federal University of Pampa, CEP 97.650-000, Itaqui, RS, Brazil
Supplemental data for this article can be accessed here.
ß 2021 Informa UK Limited, trading as Taylor & Francis Group
2 M. M. M. DAHLEH ET AL.
Introduction
Sedentarism is evidenced as one of the leading causes of
mortality globally, associated with non-transmissible dis-
eases, such as obesity and hypercholesterolemia [1]. In
this sense, physical activity is encouraged for individuals
of all ages to avoid pathologies and maintain homeosta-
sis [2]. Performing an exercise (EXE) has high therapeutic
power in systemic and psychological diseases [3].
Food compounds of natural origin can act as adju-
vants to perform an EXE protocol, as they can contrib-
ute to an increased response to these protocols [4].
Therefore, the c-oryzanol (ORY), a compound extracted
from rice bran oil, demonstrated adjuvant effects when
administering it together with EXE, like showed in a
study when thirty healthy individuals induced to ORY
supplementation, showing an intensification of anaer-
obic capacity [5]. ORY is already described in the litera-
ture for having antioxidant activity, increasing the
activity of protective enzymes in Drosophila mela-
nogaster [6]. However, no studies demonstrate the ORY
potential related to the induction of EXE given the
increase in endogenous antioxidant defenses.
Commonly, the use of murine models in EXE is veri-
fied, but there are ethical and economic limitations that
limit more extensive explorations in EXE, as well as the
number of animals used. Thus, it is necessary to use
alternative models, such as Drosophila melanogaster,
which have easy reproduction and management, pos-
sess a metabolically complex organism, preserve several
metabolic signals relevant to physical exercise [7], and
have an innate negative geotaxis mechanism, which is
used to induce continuous movements of the inverte-
brate [8,9]. The study’s main was to verify if ORY supple-
mentation improves locomotor behavior and oxidative
Figure 1. Schematic representation of the experimental EXE protocol of this study.
stress defenses in Drosophila melanogaster exposed to
the EXE protocol for seven days.
Materials and methods
Materials and fly culture conditions
The wild-type Drosophila melanogaster (Harwich strain),
obtained by CIPBIOTEC (Unipampa/S~ao Gabriel), was
used. Both sexes of flies were kept in an incubator, at a
controlled temperature (24 –25  C), humidity 30–50%,
and maintained in a twelve-hour light/dark cycle
(12:12), a standard diet consisting of corn flour, sugar,
yeast, wheat germ, powdered milk, and methylparaben.
The ORY (Figure S1) was obtained from Tokyo Chemical
Industry Co., Ltd. (Tokyo, Japan).
Experimental design and exercise protocol
Two-day-old flies were used, separated into the follow-
ing groups: Control (without EXE þ vehicle), MLF (with-
out EXE þ vehicle þ locomotor limitations), EXE
(EXE þ vehicle), ORY (without EXE þ ORY 25 lM), and
EXE þ ORY (EXE þ ORY 25 lM). EXE and EXE þ ORY flies
were subjected to the EXE protocol for seven days,
while Control, ORY, and MLF were kept in their culture
medium in the same period. On the seventh day, the
groups were subjected to behavioral tests and on the
eighth day to biochemical analyzes (Figure 1). ORY was
diluted in 1% sucrose (vehicle) with a final concentra-
tion of 25 lM [6].
Thirty flies from EXE and EXE þ ORY were submitted
to the EXE protocol (Table 1), where they were added
to a tube (6 cm high  1.5 cm diameter) and subjected
to climbing activity. The movement performed by the
 FREE RADICAL RESEARCH 3

Table 1. Exercise protocol in minutes for each day, on seven Body weight
consecutive days.
Days Exercise Rest Exercise Rest Exercise Rest Exercise Rest Total
Body weight was calculated based on the weight differ-
1 15 5 15 5 15 5 15 5 60 ence at the beginning of treatment and the end of the
2 20 5 15 5 15 5 15 5 65 seven days of the EXE protocol [11].
3 20 5 20 5 15 5 15 5 70
4 20 5 20 5 20 5 15 5 75
5 20 5 20 5 20 5 20 5 80
6 15 5 15 5 15 5 15 5 60 Sample preparation
7 20 5 15 5 15 5 15 5 65
Rest: Minutes with no movement by the machine, with the flies, kept For biochemical analysis, each group of flies was anes-
on suspension.
thetized and euthanized with the temperature at 5  C
and homogenized with HEPES buffer, pH 7.0, in 1:10
experimental apparatus consists of three rotations per
(flies/volume lL) proportion. The samples were centri-
minute (rpm), with the subtle induction of the fly’s
fuged at 1000  g at 4  C, with supernatant removed
mobility causing them to fall whenever they reach the
from the sample for further analysis.
apex of the tube, inducing their mobility by the innate
instinct of negative geotaxis (details in Figure S2, A-E).
To maintain similar conditions of manipulation of the Acetylcholinesterase (AChE) activity
animals, Control, MLF, and ORY groups were kept in fal-
con tubes during the EXE session. The experimental The supernatant was added with KPi buffer 0.25 M pH
device was developed by our research group (INPI, 8.0, 5 mM DTNB (5.50 -dithiobis (2-nitrobenzoic acid)),
BR10202000813; 2020). As the apparatus is a new with the reaction formed from the addition of 7.25 mM
device, we carried out pilot tests, demonstrating that acetylthiocholine [12]. The samples were reading with a
seven days perform a better development and adapta- spectrophotometer at 412 nm for 120 s. The AChE activ-
tion pattern to EXE in our fly model. The euthanasia of ity expressed as lmol AcSCh/h/mg protein.
the exercised flies was applied 4 h after the last EXE, all
properly fed and rested, in the same way as the other
groups. The MLF group was kept in a container with a Glucose, triglyceride levels, and glycogen
limited 5 cm of space, considered half of the other measurement
groups (10 cm) (details in Figure S3). Measurement of glucose and triglyceride levels carried
The average of all experiments using twenty flies out using LabtestV kit for the dosage of glucose and
R

was represented as n ¼ 1. Four independent experi-
ments were performed for each behavioral test or bio-
chemical analysis (n ¼ 4).
Survival rate
The survival rate was assessed by counting the number of
live flies daily until the end of the experimental period.
Open field test (OFT)
In the OFT performed to assess the flies’ exploratory
and locomotor activity, the number of quadrants per-
formed by a single fly during the 60 s period was quan-
tified [10].
Negative geotaxis
After the experiment period, fly mobility was evaluated
with the negative geotaxis method [6]. Climbing time
was assessed through the space traveled by the fly,
from the base to a limit established at the top of the
tube (8 cm high and 1.5 cm diameter).
triglyceride levels and performed according to the man-
ufacturer’s instructions. Results were obtained from a
standard glucose and triglyceride curve, with the results
expressed by mg/dL tissue.
Extraction of glycogen patterns in small tissue
amounts according to [13], in the supernatant, was
added potassium hydroxide (K(OH)) incubated at 100  C
for 10 min, after ethanol was added to the sample,
being incubated again at 70  C for 10 min, for the reac-
tion was added H2O and reactive iodine (1.5 M KI and
0.1 M I2). Reaction performed in spectrophotometry
at 460 nm.
Cell viability assay
The cell’s ability to reduce resazurin to resorufin was
analyzed, acting as a reaction fluorescence marker [14].
To form the reaction was added 180 mL of the sample,
20 mL of TRIS Buffer (pH 7.0), and 10 mL of resazurin as
the reaction catalyst. The samples were incubated on a
microplate for one hour, reading with a spectropho-
tometer at 513 nm.
4 M. M. M. DAHLEH ET AL.
Determination of reactive species (RS)
The samples were centrifugated at 3570 rpm for 5 min at
4  C. For the reaction was add 2’70 -dichlorofluorescein
diacetate (DCF-DA) used as an oxidizing agent. The fluor-
escence emission of DCF to DCF-DA monitored after 1 h
at an excitation wavelength of 488 nm and an emission
of 520 nm using a fluorescence spectrometer. The results
were obtained and expressed as a percentage of the
control DCF formation in arbitrary units (AU) [15].
Determination of thiobarbituric acid reactive
substances (TBARS)
The reaction forms a plasmatic marker oxidative stress,
malondialdehyde (MDA), as a marker of lipid
peroxidation’s final product [16]. The supernatant was
added with 20% acetic acid pH 3.5, 0.8% acid thiobarbitu-
ric (TBA) pH 3.2, 8% sodium dodecyl sulfate (SDS), being
incubated at 95  C for 120 min. Samples were submitted
to wavelength in a spectrophotometer at 532 nm, and
lipid peroxidation was expressed as MDA nmol/mg tissue.
Determination of antioxidant enzymes activity
In the superoxide dismutase (SOD) activity, the super-
natant was added with KPi buffer 0.25 M pH 8.0, N, N,
N-tetramethylethylenediamine the reaction catalyzing
by 0.15% quercetin [17]. Submit to spectrophotometer
at 406 nm for 120 s. The enzymatic activity was
expressed as U/mg protein (one unit defined as the
amount of enzyme required to inhibit the rate of oxida-
tion of quercetin by 50% at 25  C).
In the catalase (CAT) activity, the supernatant was used
with 0.25 M phosphate buffer (2.5 mM EDTA, pH 7.0), 30%
hydrogen peroxide (H2O2), and 0.012% Triton X-100, sub-
jected to spectrophotometry of 240 nm for 120 s. The
activity was expressed as U/mg protein (one unit decom-
poses 1 lmolH2O2/min at pH 7.0 and 25  C) [18].
In the glutathione-S-transferase (GST) activity, the
supernatant was added with KPi buffer 0.25 M pH 8.0,
2.5 mM EDTA, and 100 mM GSH addition, with 50 mM 1-
chloro-2–4-dinitrobenzene. Read at 340 nm spectropho-
tometer for 120 s, and expressed in million units of
enzyme activity/mg protein (mU/mg protein), the reac-
tion started with GST reduced for GSH by conjugation
reaction with 1-chloro-2–4-dinitrobenzene, forming 4-
dinitrophenyl glutathione [19].
Determination of non-protein and protein thiols
(NPSH & PSH)
To determine NPSH and PSH, the supernatant was used
to analyze protein thiol content and the pellet reserve
for NPSH measurement [20]. The supernatant was added
with 0.5 M PCA and centrifuged at 10.000 rpm for 5 min,
kept in incubation for 15 min at room temperature, with
a reading by spectrophotometer at 412 nm. Non-protein
thiols measured with the resuspension pellet of de sam-
ples was added 0.5 M Tris HCl pH 8.0, 5 mM DTNB, kept
in incubation for 15 min at room temperature, with a
reading by spectrophotometer at 412 nm.
Protein levels
The quantification of protein content was verified using
the method of [21]. Using the supernatant, distilled
water and CoomassieV brilliant blue g-250 were added
R
to the samples, and as a standard, bovine serum albu-
min was used. There were incubated for 10 min and
read through spectrophotometry at 595 nm. Protein
levels were used to quantify protein content and assess
the amount of lean mass in Drosophila melanogaster.
Statistical analysis
Normality was assessed with the Shapiro–Wilk test, and
the homogeneity of the data was analyzed using
Bartlett’s test. Then, one-way ANOVA was performed, fol-
lowed by Bonferroni’s test. The percentage of survival
determined by the Kaplan–Meier curve and the statistical
significance of log-rank Mantel–Cox. Differences between
groups considered significant when P-value <.05.
Results
Exe with ORY supplementation increase survival
rate in Drosophila melanogaster
In the assessment of survival, we observed an increase
in the survival of EXE compared to the Control
(p ¼ .0368, Figure 2) and EXE þ ORY (p ¼ .034, Figure 2),
and a decrease in on the MLF (p ¼ .0002, Figure 2). At
Figure 2. The survival rate of flies exposed to EXE protocol with
ORY in Drosophila melanogaster. The total number of flies repre-
sents the sum of four independent experiments. aDifference to
the Control group; bDifference to the MLF group.
 FREE RADICAL RESEARCH 5

Figure 3. Effect of the seven days of EXE protocol and ORY supplementation on the flies exploratory and locomotor activities in
Drosophila melanogaster. (A) Open field test; (B) Negative geotaxis test. The data are represented as the mean ± SEM. aDifference
to the Control group; bDifference to the MLF group; cDifference to the EXE group; dDifference to the ORY group.

the same time, a higher survival in the EXE (p < .0001,

Figure 2) and EXE þ ORY (p < .0001, Figure 2) groups
when compared to the MLF.

Exe improves mobility behavior with ORY

supplementation
In the OFT, the MLF group had a smaller number of
crossings than all experimental groups. There was a sig-
nificant increase in the flies’ locomotion in the EXE and
EXE þ ORY group, with a higher number of crossings
than the Control. The EXE þ ORY showed a higher num-
ber of quadrants traveled to the EXE and ORY (ANOVA:
F4.15 ¼ 69.78, p < .0001, Figure 3(A)). The negative geo-
taxis test has no statistical differences (ANOVA: F4.15 ¼
1.693, p ¼ .2039, Figure 3(B)).
Exe with ORY supplementation provide lower
levels of AChE activity
The MLF group showed a significant increase in AChE
activity concerning all experimental groups. There were
no differences in the ORY, EXE, and EXE þ ORY with the
Control (ANOVA: F4.15 ¼ 15.41; p < .0001, Figure 4).
Effect of EXE with ORY supplementation in weight
and body composition
The MLF had a significant increase in body weight com-
pared to the Control and ORY group when EXE and
EXE þ ORY significantly decreased compared to the
Control and MLF (ANOVA: F4.15 ¼ 37.93, p < .0001;
Figure 5(A)). MLF obtained a lower amount of body pro-
tein compared to the EXE þ ORY group (ANOVA: F4.15 ¼
3.676, p ¼ .0280; Figure 5(B)).
Figure 4. Effects on acetylcholinesterase activity after seven
days of EXE protocol with ORY supplementation in Drosophila
melanogaster. The data are represented as the mean ± SEM.
a
Difference to the Control group; cDifference to the EXE group;
d
Difference to the ORY group; eDifference to the EXE þ ORY.
An increase in the ratio of protein levels/body
weight was observed in groups EXE and EXE þ ORY
compared to the Control group and while it was found
a decrease in this ratio on the MLF group compared to
the EXE, ORY, and EXE þ ORY groups. ORY; EXE þ ORY
showed a higher ratio of protein levels/body weight
when compared to the ORY group (ANOVA: F4.15 ¼
19,79, p ¼ <.0001; Figure 5(C)). The MLF group had a
higher triglyceride levels/body weight ratio compared
to the Control group, while EXE þ ORY had a lower rate
of this ratio than the MLF group (ANOVA: F4.15 ¼ 5.998,
p ¼ .0043; Figure 5(D)).
Exe with ORY supplementation on lipid and
glycidic parameters of the flies
Higher levels of triglycerides were shown in MLF con-
cerning Control and ORY. EXE and EXE þ ORY flies had
6 M. M. M. DAHLEH ET AL.

Figure 5. Effects on body weight and protein body composition in Drosophila melanogaster, assessed by (A) Weight; (B) Protein
levels; (C) Protein levels/body weight ratio; (D) Triglycerides levels/body weight ratio. The data are represented as the
mean ± SEM. aDifference to the Control group; bDifference to the MLF group; dDifference to the ORY group.

a significant decrease in triglyceride levels compared to
the MLF group (ANOVA: F4.15 ¼ 5.906, p ¼ .0046; Figure
6(A)). The MLF group has a statistical difference in glu-
cose levels compared to EXE þ ORY and EXE. The EXE
group has higher glucose levels regarding Control, ORY,
and EXE þ ORY (ANOVA: F4.15¼ 11.71, p ¼ .0002; Figure
6(B)). The MLF group has lower glycogen than the
Control group. The glycogen in EXE got a significant
difference compared to Control, MLF, ORY. EXE þ ORY
had higher glycogen concerning the Control, MLF, EXE,
and ORY (ANOVA: F4.15 ¼ 37.67; p < .0001, Figure 6(C)).
Evaluation of cell viability on EXE with ORY
supplementation
The MLF group showed a significant decrease in cell
viability compared to the Control, EXE, and EXE þ ORY
(ANOVA: F4.15 ¼ 5.230; p ¼ .0077, Figure 7).
Effects of EXE with ORY on parameters of RS and
damage to the lipid components of the
cell membrane
The MLF group was observed to increase those levels
compared to all experimental groups. A significant
decrease in RS levels was showed in EXE and EXE þ ORY
compared to Control, and ORY has a significant increase
in EXE and EXE þ ORY (ANOVA: F4.15 ¼ 19.88, p < .0001;
Figure 8(A)).
In the MDA levels, MLF have an increase in those lev-
els compared to all experimental groups. EXE þ ORY
group significantly decreases these levels compared to
Control, EXE, and ORY (ANOVA F4.15 ¼ 35.06, p < .0001;
Figure 8(B)).
The response of antioxidant enzymes parameters
in EXE with ORY supplementation
SOD activity was reduced in the MLF group compared
to all experimental groups; However, the EXE flies
 FREE RADICAL RESEARCH 7

Figure 6. Effects on lipid and glycidic parameters in Drosophila melanogaster after seven days of EXE with ORY administration,
assessed by (A) Triglyceride levels; (B) Glucose levels; (C) Body glycogen reserves. The data are represented as the mean ± SEM.
a
Difference to the Control group; bDifference to the MLF group; cDifference to the EXE group; dDifference to the ORY group;
e
Difference to the EXE þ ORY group.

increase SOD defense compared to the control.

EXE þ ORY group has a significant increase in SOD
activity compared to Control, EXE, and ORY (ANOVA:
F4.15 ¼ 16.95, p < .0001; Figure 9(A)). The MLF have a
significant decrease in CAT levels regarding Control and
ORY groups. EXE was found with higher levels of CAT
activity concerning the Control group. The increase in
CAT activity was statistically significant in EXE þ ORY with
Control, EXE, and ORY (ANOVA: F4.15 ¼ 2.293, p ¼ .0006;
Figure 9(B)). GST activity has a significant increase in
EXE þ ORY compared to all experimental groups (ANOVA:
F4.15 ¼ 7.091, p ¼ .0021; Figure 9(C)).

Cell redox state after EXE with ORY

Figure 7. Evaluation of cell viability by reducing resazurin
after seven days of EXE protocol with ORY administration in
Drosophila melanogaster. The data are represented as the
mean ± SEM. aDifference to the Control group; cDifference to
the EXE; eDifference to the EXE þ ORY group.
supplementation measured by NPSH and
PSH levels
There was a significant decrease in NPSH levels in the
MLF group than Control, EXE, and EXE þ ORY groups,
8 M. M. M. DAHLEH ET AL.

Figure 8. RS levels and lipid peroxidation on Drosophila melanogaster after seven days of EXE protocol, assessed by (A) Levels of
Reactive Species; (B) Lipid peroxidation. The data are represented as the mean ± SEM. aDifference to the Control group;
c
Difference to the EXE group; dDifference to the ORY group; eDifference to the EXE þ ORY group.

Figure 9. Effects of EXE with ORY in Drosophila melanogaster on antioxidant enzymes after seven days of EXE protocol, assessed
by (A) Superoxide Dismutase (SOD) activity; (B) Catalase (CAT) activity; (C) Glutathione-S-transferase (GST) activity. The data are
represented as the mean ± SEM. aDifference to the Control group; bDifference to the MLF group; cDifference to the EXE group;
d
Difference to the ORY group; eDifference to the EXE þ ORY group.

Figure 10. Non-protein and protein thiols levels after induction of seven days of EXE and ORY administration in Drosophila mela-
nogaster, evaluated by (A) Non-protein thiols (NPSH); (B) Protein thiols (PSH). The data are represented as the mean ± SEM.
a
Difference to the Control group; cDifference to the EXE group; dDifference to the ORY group; eDifference to the
EXE þ ORY group.
while EXE significantly increased NPSH concerning
Control. ORY has lower NPSH levels regarding EXE and
EXE þ ORY (ANOVA: F4.15 ¼ 15.49, p < .0001; Figure
10(A)). It was verified a decrease in the PSH levels in
MLF compared to Control, EXE, and EXE þ ORY when
ORY has lower PSH levels than EXE and EXE þ ORY
(ANOVA: F4.15 ¼ 7.861, p ¼ .0013; Figure 10(B)).
Discussion
This study mainly showed EXE and ORY synergic effect
in improving locomotor behavior and defense against
oxidative stress in flies. Our results demonstrated that
the combination of EXE þ ORY contributed to loco-
motor activity in the flies; this is related to increased
long-term survival and one of the main factors involved
in regulating cellular processes [8,22,23]. Locomotor
activity improvement of the flies may be related to the
increase in antioxidant defense, as seen in the combin-
ation of EXE þ ORY, which may be responsible for
expanding the therapeutic action of EXE [24]. In our
climbing activity data, the flies submitted on the proto-
col showed no significant differences between the
groups, indicating no possible interference with the
innate mechanism of the negative geotaxis of flies [25].
The AChE activity in MLF represents high hydrolysis
of acetylcholine, an important neurotransmitter respon-
sible for a considerable fraction of locomotor behavior,
which may explain the decrease in locomotion of flies
in the MLF group, as in the study by [26], demonstrated
that flies that were induced to be exposed to situations
of high production of RS obtained a higher AChE, lead-
ing to less locomotion. In our study the EXE þ ORY
group, physiological levels of this enzyme were
FREE RADICAL RESEARCH 9
maintained, suggesting the preservation of loco-
motor behavior.
The results of body weight in EXE þ ORY group sug-
gest an increase in the metabolic demand for energy
substrates, changing the invertebrate’s body compos-
ition [9]. demonstrated similar effects only with EXE on
Drosophila. In addition, there is an increase in the pro-
tein levels/body weight ratio in the EXE and EXE þ ORY
groups, suggesting an increase in lean mass in
Drosophila melanogaster submitted to the EXE, similar
to that found in EXE induced mammals [24]. The MLF
group showed an increase in the triglyceride levels/
body weight ratio, predicting that the fat mass is pre-
dominant in the MLF, thus showing the importance of
the EXE performance to increase lean mass and
decrease fat mass, since EXE adjuvant to ORY was able
to decrease the triglyceride levels/body weight ratio
when compared to MLF. Therefore, the increase in body
weight of MLF in our results reflects the accumulation of
triglycerides, possibly associated with movement limita-
tion during the experiments, being a contributing factor
to the development of oxidative stress [9,22]. In contrast,
changes in the body composition of EXE and EXE þ ORY
flies, mainly with increased protein levels, can help pre-
vent oxidative damages [9,27]. The regulation of trigly-
ceride levels by EXE and EXE þ ORY indicates an efficient
adaptation of the model to EXE, since one of the charac-
teristics of the activity is the use of fat as an energetic
substrate, momentarily preserving the glycolytic reserve,
prolonging physical performance [28].
It is possible to verify an increase in glucose levels in
the Drosophila melanogaster in the EXE and EXE þ ORY
groups, when we attribute this result to the increased
need for tissue energy of the flies’ organism. The
10 M. M. M. DAHLEH ET AL.
increase in these levels may also be due to the increase
and replenishment of the animal’s body glycogen
reserves, an indicator mechanism of adaptation to EXE
by the invertebrate, considering that in mammalian
organisms, an increase in glycolytic reserves is
observed, given higher availability energy substrate,
increasing physical performance [29,30].
The adaptive response to EXE involves the favorable
disposition of the antioxidant enzymes system and the
modulation of oxidative damage [24]. Nevertheless, the
practice of strenuous EXE can increase oxidative stress
due to the production of RS [24]. The supplementation
of ORY with EXE slows down RS formation, while the
MLF group does not. Therefore, the administration of
antioxidant compounds with EXE can regulate this cel-
lular oxidative state more quickly, considering that both
act in regulating this cellular redox state [31]. The levels
of RS produced by the ORY group were similar to the
Control group, which indicates a regular production of
these components since the production of RS is natur-
ally found under physiological levels, as observed in the
study by [6]. In the MLF group, higher lipid peroxidation
was found, where research by [24] shows that the
increase in this parameter may be related to dysfunc-
tion of the transport of substances between cells,
increasing the oxidative stress, which can be even more
harmful in organisms with low antioxidant defenses, as
was verified in our data, where MLF have low activity of
these enzymes. EXE þ ORY was able to suppress lipid
peroxidation, showing that the exercised flies have a
higher capacity to control and neutralize RS, similar to
that found in murines in EXE [24,32].
The SOD and CAT activity in MLF indicates less pro-
tection against harmful quinones and semiquinones,
generated from oxygen instability conditions, wherein
[26], flies subjected to high generation of RS, showed a
decrease in the activity of these enzymes. However, in
our study, EXE and EXE þ ORY showed more significant
protection against these free radicals, suggesting a
higher antioxidant defense. Likewise, the GST activity in
the EXE þ ORY group suggests a detoxification activity
for xenobiotics in Drosophila melanogaster. In contrast,
there is less protection against possible xenobiotics in
the MLF, where [26] demonstrate that flies subjected to
conditions of high free radical production present
greater sensitivity in protecting against potential exter-
nal agents capable of generating oxidative stress, such
as xenobiotics.
The levels of NPSH and PSH presented in the MLF
group partially indicate possible deregulation in the
redox state of the cells, given its relationship with the
depletion of GSH, an endogenous antioxidant enzyme
found in large levels, which can lead to the induction of
an increase in dysfunction in cellular flies [24].
EXE þ ORY and EXE results in NPSH and PSH levels dem-
onstrate an ability to maintain the redox state of cells,
showing flies’ ability to adapt to EXE, generating
important defenses against free radicals. MLF with
decreased levels be more susceptible to deregulation of
the redox state of cells, predicting possible changes
mediated by increased oxidative stress [26,33].
The cell viability indicates that EXE þ ORY can neu-
tralize possible damage that can modify cell integrity,
unlike MLF, which can indicate cell damage, possibly
enhanced by the large generation of RS, as demon-
strated by [34], where flies subjected to situations of
high production of RS, show that the stabilization of
parameters as cell viability can be associated with cell
integrity, as well as the opposite, where less cell viabil-
ity indicates the increase in the RS, possibly causing
cell damage.
Conclusion
Our results demonstrated the ability of ORY combined
with EXE to increase the locomotor performance and
the survival of Drosophila melanogaster without over-
loading the cellular oxidative state. Likewise, it can be
seen that EXE þ ORY was able to increase antioxidant
defenses, able to control the production of RS, neces-
sary for the regulation of cellular metabolism, with
improvements in lipid and glycemic parameters, in add-
ition to the increase in the rate of protein levels, which
acts in the regulation of the organism/metabolism of
Drosophila melanogaster.
Author contributions
MMMD, SMA, MP designed the study. MMMD, SMA, VCB,
and FCP performed the biochemical analysis, MMMD, SMA,
VCB, MRP, EASM, and LBM achieve the behavioral tests.
MMMD analyze data and drafted the manuscript. SMA, VCP,
SFC, MP edited and revised the manuscript. All authors con-
tributed substantially to the research and approved the final
version of the manuscript.
Disclosure statement
The authors declare that they have no known competing for
financial interests or personal relationships.
Funding
The authors are grateful for the financial support received from
the Conselho Nacional de Desenvolvimento Cient
ıfico e

gico (CNPq) (307099/2017-2), the Fundaç~ao de Amparo
Tecnolo
 FREE RADICAL RESEARCH 11

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