Aging Brain 4 (2023) 100090

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Aging Brain
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In the quest for the ideal sweetener: Aspartame exacerbates

selected biomarkers in the fruit fly (Drosophila melanogaster)
model of Alzheimer’s disease more than sucrose
Bukola Christiana Adedayo a, b, *, Stephanie Tolulope Akinniyi a, b,
Opeyemi Babatunde Ogunsuyi b, c, Ganiyu Oboh a, b
a
Biochemistry Department, The Federal University of Technology, Akure P.M.B. 704, Nigeria
b
Drosophila Research Lab, Functional Foods and Nutraceuticals Unit, The Federal University of Technology, Akure P.M.B. 704, Nigeria
c
Department of Biomedical Technology, The Federal University of Technology, Akure P.M.B. 704, Nigeria

A B S T R A C T

This study evaluated the effect of dietary inclusions of aspartame and sucrose on some selected behavioral and biochemical indices linked with
Alzheimer’s disease in a transgenic fruit fly (Drosophila melanogaster) model expressing human amyloid precursor protein and secretase. Flies were
raised on a diet supplemented with sucrose and aspartame for 14 days. Thereafter, the flies were assessed for their survival rate, learning and
memory, as well as locomotor performance, 14 days post-treatment. This was followed by homogenising the fly heads, and the homogenates were
assayed for acetylcholinesterase and monoamine oxidase activities, as well as levels of lipid peroxidation, reactive oxygen species, and total thiol.
The results showed aspartame at all levels of dietary intake and a high proportion of sucrose significantly aggravated the mortality rate, locomotor
deficiency, and impaired biomarkers of oxidative stress and antioxidant status in the transgenic flies, while no significant effect was found on
acetylcholinesterase activity or memory function. These findings therefore suggest that while low dietary inclusions of sucrose are tolerable under
AD-like phenotypes in the flies, high inclusions of sucrose and all proportions of aspartame tested aggravated mortality rate, locomotion and
oxidative stress in the flies.

Introduction

Sweeteners are compounds that interact with the taste buds to evoke a characteristic response and enhance the perception of
sweetness. Sweeteners have the ability to impact sweet taste by masking the taste of materials to which they are added [46].
Sweeteners are categorised into two (2) categories: nutritive and artificial sweeteners. Nutritive sweeteners (NSs) contain carbohy­
drates and also provide energy (4 kcal g1 and an average of 2 kcal g1 for sugar alcohols). Nutritive sweeteners provide calories or
energy to the diet at about 4 calories per gramme. Examples of nutritive sweeteners are table sugar (sucrose), honey, etc. They are
further subdivided into monosaccharides or disaccharides, which provide 4 kcal/g, and sugar alcohols (polyols), which provide 2 kcal/
g on average[44].
Sucrose is a white sugar that contains at least 99.7% sucrose, which is a disaccharide composed of 50% glucose and 50% fructose,
and it is absolutely not necessary in our diet because it is highly refined and thus devoid of nutrients such as vitamins, minerals, and
trace elements [47]. Sugars are found naturally (intrinsic) in all fruits, vegetables, and dairy foods, or they are added (extrinsic) to
foods during processing or preparation for human consumption [48].
Sugars found in foods include glucose, fructose, galactose, sucrose, and maltose. Artificial or non-nutritive sweeteners are often

* Corresponding author at: Biochemistry Department, The Federal University of Technology, Akure P.M.B. 704, Nigeria.
E-mail address: bcadedayo@[Link] (B.C. Adedayo).

[Link]
Received 28 September 2022; Received in revised form 18 July 2023; Accepted 19 July 2023
Available online 30 July 2023
2589-9589/© 2023 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license
([Link]
B.C. Adedayo et al. Neurobiology of Aging Science 4 (2023) 100090

used as an alternative to sugar. These sweeteners are energy- and calorie-free. It is recommended that non-nutritive artificial
sweeteners (NAS) be used instead of nutritive sweeteners such as sugar to reduce overall carbohydrate and calorie intake [20].

Aspartame is an artificial sweetener that is 200 times sweeter than sucrose and is widely used as a sugar substitute in foods and beve
rages. Aspartame is a methyl ester of the dipeptides aspartic acid and phenylalanine. It is composed of substances normally found in
the diet and the body. The acceptable daily intake (ADI) is limited to 40 mg/kg/day [4]. Aspartame has a molecular formula of
C14H18N205. Aspartame has grown in popularity as a substitute for sucrose since the FDA of the United States Department of Health
and Human Services declared it safe for human consumption [7]. Aspartame is an odourless, white powder that is 200 times sweeter
than sucrose and dissolves easily in water. Aspartame has been linked to the worsening of diabetes, headaches, seizures, depression,
arthritis, and other medical problems [12].
Neurodegenerative diseases have diverse pathophysiologies with characteristic causes of cognitive impairment, behavioural
dysfunction, and memory loss, as well as speech and movement disorders[43]. Ageing, oxidative stress, DNA damage, and inflam­
mation are some of the consistent risk factors leading to neurodegenerative disease. [26]. Alzheimer’s disease is one of the neuro­
degenerative diseases and a common cause of dementia in adults. Its major symptoms are retrogressions in memory, learning, and
other cognitive functions. AD is characterised by the buildup of misfolded proteins in the brain in the form of plaques [27]. The
accumulation of beta-amyloid peptide leads to the cell death of the neurons, which causes oxidative stress and inflammation[5].
Studies have shown that consumption of sweeteners has been linked with Alzheimer’s disease and stroke.
The fruit fly (Drosophila melanogaster) is an arthropod of the family Drosophilidae and the order Diptera (two-winged insects). The
use of drosophila as a model organism in biology and medicine has a long history, including its famous use by Gregor Mendel in
studying hereditary traits [30], trait formation [31], nervous system development, and even human disease [14]. In recent years, due
to ethical concerns and calls for a reduction in the use of mammals for laboratory research, Drosophila has emerged as one of the
foremost non-vertebrate models for biological research. Studies have shown that >70% of human disease-causing genes are conserved
in drosophila[37,39].
Diet remains critical for a number of human diseases, including Alzheimer’s disease (AD). Specifically, studies have shown that
diets could produce a wide range of effects on the disease, from pathogenesis to progression and therapeutic interventions[38,36,11].
Therefore, the dietary regimen in AD is a critical factor. Sweeteners remain a key component of human diets. While caloric sweeteners
such as sucrose are popular, their use is often limited due to a number of associated health complications for prolonged use, making
non-caloric sweeteners a possible alternative; nevertheless, non-caloric sweeteners in general and aspartame in particular have not
been completely devoid of health controversies pertaining to their safety and contributions to diseases [19,16]. In this study, using the
fruit fly model of AD, we investigated the effects of aspartame and sucrose on memory index and selected biochemical parameters to
provide information on the suitability of these sweeteners under AD-like phenotypes.

2. Materials and methods

2.1. Chemicals and reagents

Aspartame was sourced from SinoSweet corporation limited (Yixing city, China), and sucrose was sourced from Symrise GmbH and
Company, Germany. Except stated otherwise, all other chemicals and reagents were of analytical grades and the water was glass distilled.

2.2. Fruit fly (Drosophila melanogaster) stock culture

Wild type D. melanogaster (Oregon strain) stock culture from the National Species Stock Centre (Bowling Green, OH, USA), was
used. The transgenic fly used (Bloomington stock P{UAS-APP695-N-myc}, P{UAS:BACE1} (BL#33798)) was obtained from University
of Drexel, Philadelphia, USA, designed by the GAL4 UAS system to express human APP and BACE-1 in their central nervous system
(otherwise referred to as Aβ fly). Bloomington stock P{GawB}elavC155 (BL#458) GAL4 line was used to drive the human APP and
BACE-1 protein expression in the nervous system of the Aβ fly as previously described [13,28,28,34]. All fly stocks were cultured as
previously described [34].

2.3. Experimental design

This study was carried out in two experiments conducted simultaneously. In the first experiment, wild-type (Harwish strain) flies,
8–10 days old, were divided into 7 groups containing 40 flies each. Group 1 was control group fed basal diet without sweetener in­
clusion. Groups 2–4 were flies fed dietary inclusions of 0.2, 0.5 and 1.0% sucrose, while groups 5–7 were flies fed dietary inclusions of
0.01, 0.02 and 0.05% aspartame. In the second experiment, group 1 was wild strain control flies fed basal diet, group 2 was transgenic
control flies fed a basal diet; groups 3–5 were transgenic flies fed dietary inclusions of 0.2, 0.5, and 1.0% sucrose; and groups 6–8 were
transgenic flies fed dietary inclusions of 0.01, 0.02, and 0.05% aspartame. Other sources of sweeteners were not included in the basal
diet. The basal diet is made up of corn meal medium containing 1% w/v brewer’s yeast and 0.08% v/w nipagin. The flies were exposed
to these treatments for 14 days, and the vials containing flies were maintained in an incubator at room temperature before being used
for different assays. The age of the flies was chosen to ensure the adult onset of dementia in the transgenic flies [33]. The choice of the
percentage inclusions of the sweeteners is based on our preliminary results (data not shown) indicating a relatively stable survial rate
in both fly strains.

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Fig. 1. Effect of Sucrose and Aspartame on Survival of a) wild type (Harwich strain) D. melanogaster; b). Transgenic APP-BACE flies *Values are
significantly different (p < 0.05) compared to a) control; b) wild control Keys: 0.2% SUC- 0.2% Sucrose, 0.5% SUC- 0.5% Sucrose, 1% SUC- 1%
Sucrose, 0.01% ASP- 0.01% Aspartame, 0.02% ASP- 0.02% Aspartame, 0.05% ASP- 0.05% Aspartame.

Fig. 2. Effect of Sucrose and Aspartame on Locomotor Performance of a) wild type (Harwich strain) D. melanogaster; b). Transgenic APP-BACE flies
*Values are significantly different (p < 0.05) compared to a) control; b) wild control #Values are significantly different (p < 0.05) compared to b)
Control Keys: As described in Fig. 1.

Fig. 3. Effect of Sucrose and Aspartame on Memory Index of a) wild type (Harwich strain) D. melanogaster; b). Transgenic APP-BACE flies *Values
are significantly different (p < 0.05) compared to wild control at 0 hr # Values are significantly different (p < 0.05) compared to wild control at 6 hr
Keys: As described in Fig. 1.

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Fig. 4. Effect of Sucrose and Aspartame on Acetylcholinesterase (AChE) Activity in a) wild type (Harwich strain) D. melanogaster; b). Transgenic
APP-BACE flies *Values are significantly different (p < 0.05) compared to a) control; b) wild control Keys: As described in Fig. 1.

Fig. 5. Effect of Sucrose and Aspartame on Monoamine oxidase (MAO) Activity in a) wild type (Harwich strain) D. melanogaster; b). Transgenic
APP-BACE flies *Values are significantly different (p < 0.05) compared to a) control; b) wild control #Values are significantly different (p < 0.05)
compared to b) Control Keys: As described in Fig. 1.

2.4. Fly survival rate assessment

In this present study, the survival rate of the wild control (WControl) flies, transgenic (Aβ) flies and Aβ flies treated with dietary
inclusions of either aspartame or sucrose were also assessed throughout the duration of the experiment. Flies were separated into six
groups as described above in the experimental design section. Following that, flies were counted every day to pool their survival rate at
the end of the experiment, which was stated as a percentage of the total fly population for each group.

2.5. Measurement of locomotor performance by negative geotaxis test

As stated previously [1], the movement of the control and treated flies was assessed using the negative geotaxis test. Prior to being
moved to the cylindrical tube for the locomotor evaluation, flies were initially immobilised in ice. Flies were tested for movement after
being immobilised by counting the proportion of those that crossed the 6 cm line after 30 s in a sterilised tube.

2.6. Memory retention assay

The flies were subjected to the aversive phototaxic suppression assay [3]as previously reported by Ogunsuyi et al., [34]. In brief,
this assay made use of a T Maze which paired the bitter taste of quinine with attraction for light. Quinine (180 µL of 1 µM) was carefully

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Fig. 6. Effect of Sucrose and Aspartame on Lipid peroxidation Level in a) wild type (Harwich strain) D. melanogaster; b). Transgenic APP-BACE flies
*Values are significantly different (p < 0.05) compared to a) control; b) wild control #Values are significantly different (p < 0.05) compared to b)
Control Keys: As described in Fig. 1.

Fig. 7. Effect of Sucrose and Aspartame on Reactive Oxygen Species (ROS) Level in a) wild type (Harwich strain) D. melanogaster; b). Transgenic
APP-BACE flies *Values are significantly different (p < 0.05) compared to a) control; b) wild control #Values are significantly different (p < 0.05)
compared to b) Control Keys: As described in Fig. 1.

applied to filter paper and placed into the lighted chamber. The phototoxic flies were allowed free access into the lighted chamber with
quinine-treated filter paper. After 45 s, the fly was tapped back to the dark chamber, and the process was repeated. Flies that failed to
walk to the lighted chamber within 45 s were scored as “Pass”. The experiment was repeated at time 0 and 6 hrs to serve as the memory
index.

2.7. Fly head tissue homogenate preparation

Experimental flies were immobilized in ice and the head rapidly dissected. The head tissue homogenate was prepared as previously
described [34]. T The total protein content of fly homogenates was determined using Bradford’s 1976 Coomassie blue method, which
used bovine serum albumin (BSA) as a standard.

2.8. Acetylcholinesterase (AChE) activity assay

The activity of acetylcholinesterase was determined according to the Ellman method [18]. The reaction mixture was made up of 10
mM potassium phosphate buffer (pH 7.4), 1.0 mM DTNB, 30 µL of homogenate, and 0.8 mM acetylthiocholine iodide. The AChE
activity was monitored at 412 nm and expressed as µmol AcSch/h/mg protein.

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Fig. 8. Effect of Sucrose and Aspartame on Total thiol Level in a) wild type (Harwich strain) D. melanogaster; b). Transgenic APP-BACE flies *Values
are significantly different (p < 0.05) compared to a) control; b) wild control #Values are significantly different (p < 0.05) compared to b) Control
Keys: As described in Fig. 1.

2.9. Monoamine oxidase (MAO) activity assay

The MAO activity was measured according to a previously reported method (Turski et al. 1973) as previously reported [33]. In
brief, the reaction mixture contained 0.025 M phosphate buffer (pH 7.0), 0.0125 M semicarbazide, 10 mM benzylamine, and 100 µL of
fly tissue homogenate After 30 min incubation, acetic acid was added and incubated for 3 min in boiling water bath followed by
centrifugation. The resulting supernatant (1 mL) was mixed with equal volume of 2, 4-dinitrophenylhydrazine, and 1.25 mL of benzene
was added after 10 min incubation at room temperature. After separating the benzene layer, it was mixed with an equal volume of 0.1
N NaOH. Alkaline layer was decanted and incubated at 80 ◦ C for 10 min. The orange–yellow color developed was measured at 450 nm
in a UV/visible spectrophotometer. The MAO activity was expressed as mmoL of amines produced per mg protein (mmoL/mg protein).

2.10. Lipid peroxidation assay

This was carried out by the method of Ohkawa et al. [35]as previously reported [33]. In brief, 0.3 mL of tissue homogenate was
added to 0.3 mL of 8.1% Sodium dodecyl sulfate (SDS), 0.5 mL HCL/acetic acid (pH = 3.4) and 0.5 mL of Thiobarbituric acid (TBA) and
the mixture was incubated at 100 ◦ C for 1 h. The resulting thiobarbituric acid reactive species (TBARS) was quantified at 532 nm in a
spectrophotometer, calculated as malondialdehyde (MDA) equivalent and expressed as µmoL of MDA produced per mg protein (µmoL/
mg protein).

2.11. Measurement of reactive oxygen species

The method described by Hayashi et al. [23], with slight modifications, was used to measure the amount of reactive oxygen species
in the fly tissues interms of H2O2 content. The reaction consists of 130 µL of sodium acetate buffer and 15 µL of tissue homogenate,
which was incubated at 37 ◦ C for 5 minutes. Thereafter, 100 µL of n-n-diethyl-para-phenylenediamine (DEPPD) reagent (a solution
consisting of 6 mg/ml of DEPPD and 4.37 µM of ferrous sulphate, dissolve in 0.1 M sodium acetate, pH 4.8) was added and the
absorbance read at 505 nm in a microplate reader. ROS levels in the fly tissues were calculated using a standard H2O2 calibration curve
and reported as Unit/mg protein, where 1 unit = 1 mM H2O2.

2.12. Determination of the total thiol content

Determination of the level of total thiol content in tissue homogenate was done by the method of Ellman [17]. The reaction mixture
was made up of a final reaction volume of 200 μL consisting of 85 μM potassium phosphate buffer (pH 7.4), 20 μL of tissue homogenate,
and 0.5 mM 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB). This was followed by 30-minute incubation at room temperature, and the
absorbance was taken at 412 nm. The total thiol content was subsequently calculated and expressed as µmol/mg protein.

2.13. Data analysis

The results of replicate readings were pooled and expressed as mean ± standard deviation (SD). One-way analysis of variance
(ANOVA) was used to analyze the results (except for the memory retention assay where two-way ANOVA was used), followed by Tukey
post hoc test, with levels of significance accepted at p < 0.05. All statistical analysis was carried out using the software Graph pad
PRISM (V.5.0).

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3. Results

The effects of dietary inclusions of sucrose (0.2–1.0%) and aspartame (0.01–0.05%) on the survival rate of both wild type (Harwich
strain) and transgenic AD model D. melanogaster are shown in Fig. 1. It was observed that in the wild-type flies, only groups
administered 0.05% aspartame exhibited a significantly reduced (p 0.05) survival rate compared to the control. However, in the
transgenic flies, there was a significantly reduced survival rate in the transgenic fly control, and the transgenic flies were administered
0.5% sucrose and aspartame (0.01–0.05%). Furthermore, the effects of dietary inclusions of sucrose and aspartame on the locomotor
performance of D. melanogaster are shown in Fig. 2. It was observed that there was no significant difference in locomotion capacity in
wild flies, while a reduction in locomotion capacity was observed in transgenic fly control and those fed dietary inclusions of the
sweeteners, which is evident by a reduction in negative geotaxis. It is important to note that transgenic flies fed dietary inclusions of
aspartame (0.01–0.05%) exhibit a further significant reduction (p 0.05) in their locomotion compared to the transgenic control flies.
Fig. 3 shows the effect of the sweeteners on the memory index of the flies. This showed that the memory index reduced significantly
in wild flies (Fig. 3a) fed dietary inclusions of 0.5% sucrose and 0.02% aspartame compared to the control at both 0 hr and 6 hr
analysis. However, in the transgenic flies (Fig. 3b), both the transgenic control flies and transgenic flies fed dietary inclusions of
glucose (0.2–1.0%) and aspartame (0.01–0.05%) exhibited significantly reduced memory index at both 0 hr and 6 hr analysis.
The effects of dietary inclusions of sucrose and aspartame on the AChE activity of D. melanogaster are shown in Fig. 4. This study
showed that in the wild flies (Fig. 4a), there was no significant difference (p > 0.05) in the AChE activity among the flies fed dietary
inclusions of both sucrose (0.1–0.5%) and aspartame (0.01–0.05%) when compared to the control. In the transgenic flies (Fig. 4b),
both the transgenic control flies and the transgenic flies fed dietary inclusions of the sweeteners exhibited significantly elevated (p
0.05) AChE activity compared to the wild control flies; nevertheless, there was no significant difference (p > 0.05) among the
transgenic flies fed the sweeteners compared to the transgenic control flies. Fig. 5 shows the effect of the sweeteners on MAO activity in
both wild and transgenic flies. In Fig. 5a, it was revealed that flies fed both sucrose (0.1 and 0.5%) and aspartame (0.01–0.05%)
exhibited significantly higher MAO activity compared to the control. Further, as shown in Fig. 4b, a similar elevated MAO activity was
observed in transgenic control flies and transgenic flies fed dietary inclusions of the sweeteners. It is also important to note that in
transgenic flies fed dietary inclusions of 0.02% and 0.05% aspartame, the MAO activity was significantly elevated compared to the
transgenic control flies.
Fig. 6a presents the effect of the sweeteners on lipid peroxidation in wild flies. This showed that aspartame at all levels of dietary
inclusion (0.01–0.05%) led to a significantly elevated lipid peroxidation level when compared to the control. On the contrary, for
dietary inclusion of sucrose, a significantly elevated lipid peroxidation level was only observed in flies with dietary inclusion of 0.5%
sucrose. In the transgenic AD fly model, there was no significant difference between the wild control flies and the transgenic control
flies in terms of their lipid peroxidation levels (Fig. 5b). Nevertheless, transgenic flies fed all levels of dietary inclusions of aspartame
(0.01–0.05%) exhibited significantly higher lipid peroxidation levels when compared to the transgenic control flies. A similar sig­
nificant elevation in the levels of lipid peroxidation was only observed in the transgenic flies fed dietary inclusions of 0.5% and 1.0%
sucrose when compared to the transgenic control flies. The results obtained for the effects of the sweeteners on reactive oxygen species
(ROS) levels in wild (Fig. 7a) and transgenic (Fig. 7b) flies are quite similar to those obtained for lipid peroxidation as earlier described.
However, in the transgenic flies, significant elevations in the ROS level were observed for all levels of aspartame diet inclusions
(0.01–0.05%) and dietary inclusions of 1.0% sucrose only.
The total thiol content showed that for the wild-type flies (Fig. 8a), dietary inclusions of sucrose (0.2% and 0.5%) and aspartame
(0.01% and 0.02%) led to a significant reduction in the total thiol contents. Further, in the transgenic flies (Fig. 8b), there was a
significant reduction in the total thiol content in the transgenic control flies compared to the wild control flies. In addition, transgenic
flies fed dietary inclusions of sucrose (0.2–1.0%) and aspartame (0.01–0.05%) also exhibited significantly reduced total thiol contents
compared to the transgenic control [Link]. 8.

4. Discussion

Alzheimer’s disease commonly involves decline in cognitive function, which begins to appear as memory loss[15]. In this study, we
confirmed this AD-like phenotype in the transgenic flies, resulting in their significantly reduced memory index compared to the wild-
type flies. The use of the aversive phototaxis suppression assay to monitor learning and memory in flies has been well reported
[3,2,33,21]. The findings of this study also agree with our earlier findings on reduced memory index in these transgenic fly models
[32,34]. Furthermore, similar neurodegenerative phenotypes have been reported by Greeve et al. [22] in triple transgenic flies
expressing human APP (hAPP), human β-secretase (hBACE), and Drosophila -secretase presenilin (dPsn). Also, a memory decline was
reported in flies expressing human A42 [24]. Similarly, the fact that all the transgenic flies fed dietary inclusions of both aspartame and
sucrose also exhibited a significantly reduced memory index compared to the wild-type flies further shows that the loss in memory
conditions is preserved but not aggravated.
Considering the pivotal role the cholinergic system plays in learning and memory, and specifically AChE being a therapeutic target
in AD, AChE activity was monitored in the fly heads to support the observed memory phenotype. This finding corresponds with the
acetylcholinesterase activity in the fly head, in which, while all the transgenic fly groups exhibited significantly elevated AChE activity,
there was no significant difference between AChE activity in the transgenic control flies and transgenic flies fed dietary inclusions of
both aspartame and sucrose. This, therefore, could help explain the possible mechanism behind the observed effects on the memory
index. On the contrary, in wild-type flies, flies fed dietary inclusions of glucose at 0.5% and aspartame at 0.02% exhibited a significant
decline in memory index. Nevertheless, this does not correspond with the observed head AChE activity, in which no significant

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difference was observed among the treatment groups. While the reason for these observations might not be fully understood at this
level, it has given information to suggest the involvement of the sweeteners more in pathogens than the progression of the pathological
phenotypes. Also, it suggests the involvement of other metabolic pathways in the observed findings.
Cognitive dysfunction and AD are multi-etiological diseases involving several metabolic and signalling pathways and pathological
indices. To further investigate these in this study, investigations were made into the effects of the sweeteners on markers of oxidative
stress and redox homeostasis. The observed significant elevation in lipid peroxidation level in the wild-type flies fed dietary inclusion
of sucrose at the highest percentage inclusion of 1.0% and all ranges of aspartame inclusion (0.01–0.05%) suggest that the sweeteners
elicit redox imbalance through the generation of free radical-mediated oxidative stress. This was further substantiated by a corre­
sponding finding obtained for the elevated levels of reactive oxygen species (ROS) and reduced total thiol content. ROS are known to
be a major cause of redox imbalance by eliciting oxidative stress chain reactions, including the oxidation of macromolecules, as
observed in lipid peroxidation, protein carboxylation, and DNA damage [42]. These could therefore suggest that these sweeteners, at
these percentage inclusions, induce ROS-mediated oxidative stress and lipid peroxidation in the fly head. Ashok and Sheeladevi [6]
had earlier reported that aspartame (although at a chronic administration of 40 mg/kg b.w.) elicits oxidative stress and neurode­
generative apoptosis in rat brains. It is therefore not surprising to see similar effects in the transgenic flies. The observed significant
elevation of lipid peroxidation and ROS levels in transgenic flies fed dietary inclusions of 1.0% sucrose and aspartame (0.01–0.05%)
compared to the transgenic flies suggests that the sweeteners at these proportions further aggravated oxidative stress and redox
imbalance in the flies. This is very critical considering the role oxidative stress plays in the progression of AD and dementia.
Previous studies have shown a direct link between free radical-mediated oxidative stress and the pathogens and progression of AD
[9,10]. Consequently, antioxidant-based therapies, either through augmentation of endogenous antioxidants or dietary intervention,
have been well justified for the prevention and management of AD and dementia [40]. Thiols serve as a major source of endogenous
antioxidants for mitigating the deleterious effects of free radicals. The significant reduction in total thiol content, particularly in
transgenic flies fed dietary inclusions of both sweeteners compared to the transgenic control group, could further support the
aggravation of redox imbalance and oxidative stress in the flies.
The impairment in monoamine oxidase (MAO) activity in both wild-type and transgenic flies fed dietary inclusions of both
sweeteners could further support the mechanism of dysregulation in redox homeostasis by the sweeteners in both the wild and
transgenic flies. Studies have shown that elevated MAO activity contributes to the generation of ROS, which are byproducts of their
enzymatic activity on biogenic amines [25]. Consequently, the significantly elevated MAO activity observed, particularly in transgenic
flies fed dietary inclusions of aspartame (0.02 and 0.05%), could have contributed to the aggravated level of oxidative stress in the flies
when compared to the transgenic control group.
Furthermore, MAO is critical to the monoamine neurotransmitter systems, particularly dopamine-mediated neurotransmission,
and have been implicated in different neurodegenerative diseases, including those involving cognitive dysfunction like AD [41,8] and
neuromuscular dysfunction like PD [45]. In this study, we observed in the transgenic flies a significant reduction in their locomotor
performance. Particularly in flies fed aspartame at all proportions of inclusions, a significantly reduced locomotor performance was
observed, suggesting a further aggravative effect of this sweetener in the AD fly model.
Put together, the elevated markers of oxidative stress (lipid peroxidation, ROS, and MAO), coupled with the reduction in endog­
enous antioxidants (total thiol) in transgenic flies fed dietary inclusions of both sucrose and aspartame, could provide mechanisms to
support a state of aggravated redox imbalance and oxidative stress in the flies. Considering the role of oxidative stress in cell viability
and toxicity, it comes as no surprise that this group of flies also experienced an aggravated mortality rate. Nevertheless, the reduced
memory index in the transgenic flies was not significantly aggravated in the transgenic flies fed dietary inclusions of both sweeteners.

5. Conclusion

This study has shown that dietary inclusions of aspartame and sucrose aggravated the elevated markers of oxidative stress and
reduced endogenous antioxidants in both wild-type and transgenic AD fruit fly (Drosophila melanogaster) models. These aggravated
effects were most profound in flies fed dietary inclusions of aspartame (0.01–0.05%) and 1.0% sucrose. However, we found no evi­
dence of aggravation in the impaired memory function but for the locomotor performance in these flies. This could therefore provide
evidence to support the idea that aspartame and a high proportion of sucrose could aggravate AD-like phenotypes in the fruit fly model.
Further studies are, however, encouraged to provide more justifications for these findings.
Practical application.
Sweeteners are a key component of human diets. While caloric sweeteners such as sucrose are popular, their use is often
discouraged due to a number of associated health risks, particularly for prolonged use, making non-caloric sweeteners possible al­
ternatives; nevertheless, non-caloric sweeteners in general and aspartame in particular have not been completely devoid of health
controversies relating to their safety and contributions to diseases. In this study, using the fruit fly model of AD, we investigated the
effects of aspartame and sucrose on survival rate, memory index and selected biochemical parameters to provide information on the
suitability of these sweeteners under experimental AD-like phenotypes.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to
influence the work reported in this paper.

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Acknowledgements

The authors appreciate Dr. Daniel R. Marenda of the Department of Biology, University of Drexel, Philadelphia, USA, for the
generous donations of the transgenic files used for this study.

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