Glycative Stress Research
Online edition : ISSN 2188-3610
Print edition : ISSN 2188-3602
Received : April 1, 2020
Accepted : April 20, 2020
Published online : June 30, 2020
doi:10.24659/gsr.7. 2_132

Original article

Actions of various sweeteners on rat sperm.
Yukiko Minamiyama † 1), Keiko Kobayashi 1), Yuta Noguchi 2), Shigekazu Takemura 3), Hiroshi Ichikawa † 2)
1) Food Hygiene and Environmental Health Division of Applied Life Science,
Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Kyoto, Japan.
2) Department of Medical System Protective Health and Medicine Laboratory,
Graduate School of Life and Medical Sciences Doshisha University, Kyotanabe, Kyoto, Japan.
3) Department of Hepato-Biliary-Pancreatic Surgery, Graduate School of Medicine, Osaka City University, Osaka, Japan† This author contributed equally to this work.

Abstract

Purpose: Sweeteners other than sugar are often used in foods aimed at reducing calories. However, it has been reported thatsome of these sweeteners exhibit reproductive toxicity such as sperm count reduction and decreased sperm motility caused by
the involvement of reactive oxygen species (ROS). In this study, we evaluated the actions of sucrose (Suc), sucralose (SCL),
aspartame (APM), or neotame (NTM), which are food additive sweeteners frequently consumed on a daily basis, on rat spermin vitro. In addition, rats were given APM and NTM to examine their actions on sperm.
Methods: The animals used were Wistar male rats aged 12 to 15 weeks. Spermatozoa were removed from the epididymis
and subjected to experiments. In the in vitro experiment, a stock solution of TYH medium containing sperm was prepared,
and various concentrations of additives were added; after 5 min, the sperm motility was measured by a sperm motility
analysis system (SMAS). Also, sperm stock solution containing sperm was diluted 5- fold, and L-012 (100 µM) was added asa fluorescent probe of ROS, followed by measuring the luminescence intensity by the chemiluminescence method. In in vivo
studies, APM (250 mg/kg/day) or NTM (40 or 100 mg/kg/day) below the NOAEL (no observable adverse effect level) was
administered as a mixed diet; after 2, 4, and 8 weeks, blood and epididymis were collected. As in the in vitro study, sperm
motility and ROS production were measured, and the organs were analyzed for oxidative stress-related proteins.
< 1 µM) or APM
Results: In In vitro tests, ROS production had little effect on the addition of Suc ( =< 500 µM), SCL ( =(<= 10 µM), while there was a tendency for NTM (1 µM) to decrease compared to the control group. Impacts were negligible<<on sperm motility by the addition of Suc ( <= 5 mM), SCL ( = 10 µM), or APM ( = 100 µM), however, sperm motility was>significantly reduced by the addition of NTM ( = 0.1 µM). In in vivo tests, APM administration did not change the sperm
count, but ROS production was significantly increased at 2 weeks compared to the control group; at 8 weeks, the total
sperm motility showed a declining trend. NTM significantly increased ROS production after 4 weeks of administration,and decreased sperm linear velocity, curve velocity, head amplitude, and head frequency at 8 weeks; the sperm count andtotal sperm motility were not affected; in parallel with the increase in ROS production, NTM increased the amount of lipid
peroxidation-modified protein with positive 4-hydroxy-2-nonenal (4 -HNE) in the cauda epididymis.
Conclusion: Some sweeteners affected sperm where the administration of APM and NTM increased ROS production prior
to sperm dysfunction. This mechanism could be caused by abnormalities in the electron transport system of mitochondria andoxidative stress due to decreased antioxidant capacity.

KEY WORDS: sweeteners, oxidative stress, sperm motility, reactive oxygen production

Correspondence to: Professor Yukiko Minamiyama
Food Hygiene and Environmental Health Division of Applied Life Science,
Graduate School of Life and Environmental Sciences, Kyoto Prefectural University.
1- 5 Shimogamo Hangi-cho, Sakyo-ku, Kyoto 606 -8522, Japan
TEL: +81-75-703-5409 e-mail: yukiko-m@kpu.ac.jp

Co-authors: Kobayashi K, k-kobayashi@kpu.ac. jp; Noguchi Y, uta090226@gmail.com;
Takemura S, takemura@med.osaka-cu.ac.jp; Ichikawa H, hichikaw@mail.doshisha.ac.jp_ 132 _

Glycative Stress Research 2020; 7 ( 2) : 132-141(c) Society for Glycative Stress ResearchGlycative Stress Research

Introduction
With the expansion of the convenience store, fast foodand restaurant industries, there are concerns about the
influence of food additives. This reflects the Japanese social
situation, such as aging society, working together, and single
families. Concurrently, import / export and transportation offood from the production area are frequent, and an increasein food mileage is also a problem. For this reason, many efforts
have been made to prevent food poisoning and the deteriorationof foods. Typical examples include food additives, insecticides,
preservatives, fungicides, and pesticides. Many chemicals areused in these and potential endocrine disrupting substances(environmental hormones) are considered to have in vivo
adverse actions. It is reported that they act on reproductive
toxicities such as genital abnormalities and reduced
conception rate 1) and on the developmental brain, thus
deteriorating learning ability and social recognition 2, 3). A
representative example of social properties is bisphenol
A (BPA), which is used as a plastic plasticizer for foodcontainers. BPA are shown to cause elevated reactive oxygen
species (ROS) production due to reduced antioxidant enzyme
activity and manifest reproductive toxicity such as reduced
sperm motility and sperm count in rats 4, 5). Furthermore,diethylstilbestrol (DES), which was once used as an abortion
inhibitor, is reported to reduce sperm count and sperm
motility in humans 6). Also, some daily food additives havereproductive toxicity 7).
In recent years, the growth of the diabetic population
is noted and an increasing number of people use artificial
sweeteners for the purpose of calorie restriction.
Sucrose, a natural sweetener, is a major componentof sugar and is one of the major sweeteners in developed
countries. However, since 2002, the WHO (World HealthOrganization) has been monitoring daily sugar intake for the
purpose of preventing lifestyle-related diseases and dentala)CH 2OHH

CH 2OH
H
O
H HO

O H
OH H
O

OH
H

caries, and recommended sugar should comprise less than
10 % of daily energy intake. Furthermore, a new guideline in
March 2014 was issued to reduce the amount to less than 5%(about 25 g) from 2020. These guidelines imply that adults
are restricted to about six teaspoons of sugar a day. This limit
is limited to a very small intake given that a teaspoonful of
tomato ketchup contains about 4 g of sugar and a carbonateddrink contains about 40 g of sugar. Given that a teaspoon of
tomato ketchup contains about 4 g of sugar and a carbonateddrink contains about 40 g of sugar, it turns out that it isrestricted to very small intake. Therefore, concerning the factthat the utilization rate of artificial sweeteners will increase,
it is necessary to carefully evaluate the safety. In this study,
we investigated the adverse actions of typical sweeteners,
sucrose (Suc), sucralose (SCL), aspartame (APM) and neotame(NTM) on rat sperm. In addition, sperm contains a large
amount of polyunsaturated fatty acids in the cell membrane,
and thus is susceptible to oxidative damage and is said to be
the most sensitive cell in a living body to external stimuli 8).
Therefore, when using this, the safety evaluation may be an
excellent high-sensitivity safety test.
The artificial sweeteners, SCL, APM, and NTM areabout 600 fold, 200 fold, and 10,000 fold more sweet thanSuc, respectively 9-11), which are recently used frequently
because the amount needed to feel the same sweetness
is less than Suc and lower in calories. However, many
artificial sweeteners have been questioned for their safety.
For example, APM is absorbed from the intestinal lumen
upon ingestion and is metabolized to phenylalanine, aspartic
acid, and methanol; methanol produces ROS, causing liver
damage; increased phenylalanine may affect brain dopamineand norepinephrine levels, thus modifying brain function 12-14).
NTM, on the other hand, is a dipeptide methyl ester derivativesynthesized by reductive N-alkylation of APM and has asimilar chemical structure to APM (Fig. 1). Since NTM is nothydrolyzed to phenylalanine and aspartic acid, it can be usedb)

CH 2OHOH

CH 2OH
O
CI
H
OH H
H

OH H

H

c)

O

CI H 2C
O

OH

H

H

HO

OH

H

CH 2CI

d)
HO

O
O

OCH 3

N
H
OH

NH 2

H
NH

O

O
N
H

H 3C

O

H 3C

CH 3

Fig. 1. Structural formula of sweeteners.
a) Sucrose (Suc). b) Sucralose (SLC). c) Aspartame (APM). d) Neotame (NTM).

_ 133 _

H O
O

CH 3

Actions of Various Sweeteners on Rat Spermfor patients with phenylketonuria, and its usage is expected toincrease.
In 1992, Dr. Carlsen and his colleagues in Denmark
reported that sperm counts and semen volume in men from
20 countries have declined by almost half in 50 years 15),
suggesting that, as the cause, the influence of food additives
and environmental hormones are present. Therefore, it isimportant to examine the presence or absence of sperm
toxicity as a safety evaluation of food additives. In this study,
we verified whether or not sperm toxicity is present andinvestigated the mechanism, using Suc, SCL, APM and NTM
in the in vitro experiments and the rats administered APM
and NTM.

Methods
Materials.

Reagents
APM and NTM were provided by AstaReal Co., Ltd.
(Minato-ku, Tokyo, Japan). Other reagents were of biochemical
grade.
Animals
In this experiment, male Wistar rats (Clea Japan, Tokyo,
Japan) of 12 to 15 weeks of age were used. CE-2 (Clea Japan)
was used as a standard feed for the control group in the invivo experiment. APM and NTM were mixed and fed freely
at 250 mg/kg/day, 40 or 100 mg/kg/day, respectively. Water
was freely available in all groups. After breeding for 2, 4 or 8
weeks, the rats were anesthetized by intraperitoneal injectionof 5 g/kg of 25% ethyl carbamate (urethane) (Wako Pure
Chemical, Osaka, Japan), and perfused with 0. 9 % saline atroom temperature after blood collection from abdominal aorta.
After sacrifice, blood, epididymis, and testes were collected
for analysis. The animal experiments were performed with theapproval of the Animal Experiment Committee of Doshisha
University.

.

Radical scavenging ability in plasma
Each radical was measured using an X-band Microwave
Unit ESR device (RE Series: JEOL, Tokyo, Japan) according
to a previous report 16). For analysis of each radical, WINRAD (Version 1.30: Radical Research Inc, Tokyo, Japan) wasused.

Sperm function
A 2-mm incision was made on the surface of the cauda
epididymis with scissors, and the sperm was extruded bypinching. The sperm obtained was collected with the tip oftweezers and placed in 1.5 mL of TYH medium (Mitsubishi
Chemical Medience Co., Tokyo, Japan) preheated in a CO 2
incubator at 37 °C for 3 hours or more, followed by slow
tapping after 5-min incubation, thus preparing a stock
solution of TYH medium containing sperm.
In the in vitro experiment, the sperm stock solution
was diluted 5-fold with TYH at 37 °C, to 200 µL of which
2 µL of a 100-fold concentration of each food additive
was added, and 5 min later, put 4 µL of this solution into

a dedicated chamber plate (SC20-01-04-B: Leja, NieuwVennep, Netherlands). Using a sperm movement analysis
system (SMAS; Detect, Tokyo, Japan), sperm motility (%),
straight velocity (µm/sec), curve velocity (µm/sec), straightness(straight velocity / curve velocity), head amplitude (µm), andhead frequency (Hz) were measured.

ROS production from sperm
The sperm stock solution prepared above was diluted 5
fold with TYH medium warmed in a CO 2 incubator at 37 °C
for 3 hours or more to prepare a 5-fold diluted sperm solution,
5 µL of which was placed in 945 µl of DMEM medium(D6046: Sigma, Osaka, Japan), and the luminescenceintensity was measured by the chemiluminescence method(AccuFLEX Lumi400; Hitachi Healthcare Systems, Tokyo,
Japan).
Thirty seconds after the start, 50 µL (final concentration
100 µM) of 2 mM L012 (Wako Pure Chemical) as a Luminalbased chemiluminescent probe, after 100 sec, 10 µL of each
additive solution, and after 240 sec, 5 µL (final concentration
100 U/mL) of the prepared superoxide dismutase (SOD:
Nippon Kayaku, Tokyo, Japan) solution readjusted to 10,000
U/mL was added. The amount of ROS production was
evaluated based on the peak emission intensity.
The sperm count in the in vivo experiment wasdetermined by cutting the left cauda epididymis and filtering
the impurities using a 70 µm cell strainer (REF352350:
BD Falcon, NJ, USA) and placing it in 50 mL Falcon. Thissolution was diluted 20-fold with phosphate buffer saline,
PBS (−), and the sperm number was counted using a cellcounter. Then, the total number of sperm contained in 1. 5
mL of TYH medium was calculated.

Western blot analysis
The cytosol of the cauda epididymis was mixed withRIPA buffer so that the volume became 9 times that of theorgan, and homogenized with a homogenizer (NS-360D:
Microtech Nition, Chiba, Japan) at 20 krpm for 30 sec on
ice. Thereafter, it was centrifuged at 4 °C, 12,000 rpm
for 10 min in a centrifuge (CT15RE: Hitachi Healthcare
Systems, Tokyo, Japan). The supernatant was collected in
1.5 mL Eppendorf tubes and used as an organ cytosol stock
solution. The appropriate amount of protein was subjected
to SDS-PAGE (sodium dodecyl sulfate - polyacrylamide gelelectrophoresis). As a molecular weight marker, a pre-stained
protein marker (Wako Pure Chemical) was used.
After adding the primary antibody, anti-HNEJ-2
antibody (JaICA, Shizuoka, Japan), diluted 1: 5000 with
Canget Signal Solution 1 (Toyobo, Osaka, Japan), thespecimen was sealed to prevent air bubbles, stored at 4 °C,
and reacted overnight. After the overnight reaction, the plate
was immersed in TBS-T (Tris buffered saline with Tween20) and shaken at room temperature for 7 min with a shaker,
followed by replacement of the solution. This procedure
was repeated three times. Next, the membrane is placed ina hybridization bag, and a secondary antibody (Dako Japan,
Kyoto, Japan) diluted 1: 20,000 with Canget Signal Solution
2 is added and sealed without air bubbles, followed byreaction at room temperature for 90 min. After the reaction,_ 13 4 _Glycative Stress Research

the membrane was immersed in TBS-T and shaken witha shaker for 7 min. This was repeated three times, then,after washing, the membrane was immersed in the coloring
solution for 2 min, followed by the detection of bands with
Image Quant Las 4000 mini (GE Healthcare Lifescience,
Buckinghamshire, UK).

Membrane potential of sperm mitochondria
The potential difference of mitochondria was measured
using JC-1 Mitochondrial Membrane Potential Detection kit(Biotum, CA, USA). A stock solution was prepared in a
TYH medium so as to have a cell solution (1 × 10 6 cells/mL).
And then a NTM solution was added, and the mixture wasincubated at 37 °C in a CO 2 incubator for 30 min. Then,the mixture was centrifuged at 400 x g for 5 min at room
temperature, removed the supernatant, added 500 µL of
JC-1 reagent diluted 1 : 10 with assay buffer, and incubatedat 37 °C in a CO 2 incubator for 15 min. The mixture wascentrifuged at 400 x g for 5 min at room temperature. Afterremoving the supernatant, 2 mL assay buffer was added
and centrifuged at 400 x g for 5 min at room temperature.
Repeatedly 300 µL of assay buffer were added, then 100 µL
was dispensed per well in a 96-well black plate (NUNC),
followed by the measurement using a fluorescent plate-reader(live cells in red: excitation [Ex] 560 nm, emission [Em]595 nm, apoptosis-inducing cells in green: Ex 485 nm, Em
535 nm).

Statistical analysis
A software, SPSS ver.22 (IBM Japan, Tokyo, Japan),
was used for statistical analysis of data. Using the one-way
analysis of variance's Turkey test, p < 0.05 was defined as
the significance level.

Results

In vitro actions of food additives on sperm
Addition of Suc, SCL or APM had no effect on ROS
production (Fig. 2). The addition of NTM tended to rise ROS
production below 10 nM, while, at higher concentrations,
it tended to decrease. Addition of Suc, SCL or APM had
almost no effect on sperm motility, which, in contrast,
was significantly reduced by the addition of NTM with aconcentration above 0.1 μM (Fig. 3 ). Additives did not affectsperm linear velocity, curve velocity straightness, head
amplitude, or head frequency. The decrease in sperm motility
by NTM correlated with a drop in mitochondrial membranepotential (Fig. 4 ).

a)

b)

c)

d)

Fig. 2. ROS production from sperm by addition of sweeteners.
a) Sucrose (Suc). b) Sucralose (SLC). c) Aspartame (APM). d) Neotame (NTM). Results are expressed as mean values ± SE,
n = 4 ~ 8. ROS, reactive oxygen species; SE, standard error.

_ 135 _

Actions of Various Sweeteners on Rat Sperm

In vivo actions of APM and NTM
There was no significant difference in food intake andbody weight between APM and NTM administration. In
addition, the free radical scavenging activity of plasma was
reduced by the administration of both additives for someradical species, and particularly after 2 weeks, the scavenging.
activities for OH and O 2 − were reduced (Fig. 5 ).

.

ROS production from sperm and expression oflipid peroxide-modified protein by APM and NTM
administration
At 2 weeks of APM administration, ROS production
significantly rose compared to the control group (Fig. 6-a),while, at 8 weeks, it was not significantly different from thatof the control. Two weeks after administration of NTM (40mg/kg/day), ROS production was not changed compared tothe control group, while it rose at 4 and 8 weeks (Fig. 6-a).
In the testis and cauda epididymis, bands of 4-HNEmodified protein were detected at 53 kDa and 28 kDa. Since

the same shading tendency was observed in the bands ofall organs, Fig. 6-b shows a band of 53 kDa in the cauda
epididymis. Expression of HNE-modified protein was
significantly elevated 2 weeks after APM administration. At
4 and 8 weeks of NTM (40 mg/kg/day) administration, theexpression of HNE-modified protein was elevated comparedto the control group, becoming significant at 4 weeks.

Actions on sperm function
Administration of APM and NTM did not change sperm
count or total sperm motility (Fig. 7 ). APM administration did
not change sperm straightness, head amplitude, curve speed,or straight speed. Figure 8 shows the sperm straightness,head amplitude, and head frequency of the APM or NTM
administration group when the control group was definedas 100 %. Eight weeks after NTM administration, the sperm
straightness showed an upward trend, and the head amplitudeand head frequency decreased significantly, indicating thepossibility of reduced fertility.

a)

b)

c)

d)

Fig. 3. Actions of sweetener on sperm motility.
a) Sucrose (Suc). b) Sucralose (SLC). c) Aspartame (APM). d) Neotame (NTM). Results are expressed as meanvalues ± SE, n = 4 ~ 8, * p < 0.05 vs control without sweeteners by Turkey test.

_ 13 6 _Glycative Stress Research

Fig. 4. Decrease actions of NTM on the membrane potential of sperm mitochondria.

NTM was added to the sperm suspension and incubated for 30 min. Results are expressed
as mean values ± SE, n = 6, * p < 0.05 vs control without sweeteners by Turkey test. NTM,
neotame; SE, standard error.

a)

1O

b)

2W
. OH

8W
. OH

150

150

.

1O

50

0

0

RO

ROO

.

.

O2 -

2

50

. CH3

.

100

O2 -

100

2

. CH3

RO

：CT L
：APM

ROO

.

.

：NTM

Fig. 5. Radar chart of plasma free radical scavenging activity after administration of APM and NTM.
a) At 2 weeks. b) At 8 weeks. Results are expressed as values when ROS elimination rate in control plasma definedas 100%. APM, aspartame; NTM, neotame; ROS, reactive oxygen species.

_ 137 _

Actions of Various Sweeteners on Rat Sperm

a)

b)

c)

Fig. 6. ROS production from sperm and 4-HNE-modified protein in the cauda epididymis induced
by administration of APM and NTM.
a) ROS production. b) 4-HNE-modified protein (53 kDa). c) Membrane image by Coomassie brilliant blue

staining. Results are expressed as mean values ± SE, n = 3 ~ 5, * p < 0.05 vs control by Turkey test. APM,
aspartame; NTM, neotame; ROS, reactive oxygen species; 4-HNE, 4-hydroxy-2-nonenal; SE, standard error.

_ 13 8 _Glycative Stress Research

Fig. 7. Actions of APM and NTM
administration on total motile
sperm count.

The sperm motility was measured 2,
4 or 8 weeks after administration of
APM (250 mg/kg/day) or NTM (40 or
100 mg/kg/day). Results are expressed
as mean values ± SE, n = 3 ~ 5. APM,
a spa r t a me; N T M, ne ot a me; SE,standard error.

a)

b)

c)

Fig. 8. Effects of APM or NTM administration on
sperm motility.
a) Straightness. b) Head amplitude. c) Head frequency.

Results are expressed as mean values ± SE, n = 3 ~ 5,
* p < 0.05, ** p < 0.01 vs control by Turkey test. APM,
aspartame; NTM, neotame; SE, standard error.

_ 13 9 _Actions of Various Sweeteners on Rat Sperm

Discussion
In the present study, we investigated the actions ofsweeteners on sperm in Wistar rats in vitro and in vivo. In thein vitro study, sperm motility was not affected by the additionof Suc or artificial sweeteners, SCL and APM, and no sperm
toxicity was noted.
In contrast, a significant decrease in sperm motility anda rise in ROS production were observed with the additionof NTM. Furthermore, the sperm mitochondrial membranepotential dropped significantly at this time.
On the other hand, in vivo studies showed that sperm
count was not changed by APM administration, while thetotal sperm motility showed a declining trend over 8 weeks.
NTM administration did not change sperm count or total
sperm motility, but increased sperm straightness, straightvelocity, curve velocity, head amplitude, and head frequency,
which are important for fertilization function. Furthermore,
ROS production from sperm has been shown to increase ROS
production prior to such a decline in sperm function. Thesefindings are similar to the results of the administration testof endocrine disruptors such as bisphenol A in the previous
report 17).
Sperm midpieces are abundant in mitochondria
which produce ATP as an energy source for movement,
concurrently, ROS is produced from the electron transportsystem. Therefore, when this ROS is overproduced, it causes
leakage from mitochondria, resulting in impairment of sperm
function and declined motility. This study has revealed
that the administration of APM and NTM increased ROS
production and HNE-modified protein, which is a secondary
product of oxidation of ω6-polyunsaturated fatty acids.
From the above, it was considered that the sperm membrane
was oxidized due to the elevation in oxidative stress, which isone of the causes of the decline in sperm function.
SCL, which showed no sperm toxicity in vitro, has astructure where chlorine is attached to the molecule, andits safety has been questioned as a type of organochloride.
Although some reports indicate that it is non-toxic and does
not affect reproduction in gene expression 18, 19), considering
the actions of metabolites, it may be necessary to study interms of oxidative stress in vivo.

impaired glucose tolerance 20). In recent years, analyses
of more than 4,000 people have identified that drinking
soft drinks with artificial sweeteners is associated with anincreased risk of stroke and dementia. The observational
studies show that, in those who drink artificially sweetened
beverages at least once a day, the risk of ischemic stroke
almost triples and Alzheimer's risk is 2.9 times higher
over 10 years, compared with those who do not 21). They
are correlated, however, it is not clear that the causes andeffects may all correspond, and further tracking is needed.
Furthermore, a meta-analysis of the ingestion sweeteners
other than sugar has been reported to affect health, but noclear results have yet been obtained 22).
In this study, it was revealed that administration below
the maximum non-toxic level caused a decline in the in vivo
antioxidant capacity, an elevation in ROS production from
sperm, and actions on sperm motility. It is also importantto re-evaluate the previous toxicity tests. In addition, theconcept of exposome has been proposed for all chemicals
other than genetic factors that are exposed throughout life 23, 24).
Exposome widely includes extrinsic factors such as radiation
as well as food additives and intrinsic factors such asoxidative stress and inflammation, where the association
with inflammatory bowel disease 25) and various diseases has
been recognized 26-28). Conversely, transcriptome analysis hasreported that dietary intervention can prevent exposomerelated diseases 29). Based on these concepts, further researchon disease prevention and health maintenance is essential.

Conclusion
In the present study, some sweeteners have been revealed
to have sperm toxicity due to oxidative stress.

Conflict of Interest Statement

Currently, in Japan, the ADI (acceptable daily intake)of food additives is set as NOAEL (maximum non-toxic levelin animal tests) x 1/100, with a safety factor of 100. The
NOAELs for APM and NTM are 4,000 mg/kg/day and 96. 5
mg/kg/day in rats, respectively. In contrast, spermatozoa
toxicity was suggested in this intervention experiment, eventhough it was examined at low concentrations at 250 mg/kg/
day in APM and 4 0 mg/kg/day in NTM, which were lower
than NOAEL. According to a survey by the Ministry of
Health, Labor and Welfare in 2011, the daily intake of APM
was reported to be 0.019 mg/kg/day in humans. The toxicityof APM alone may be negligible, however, the actions oflong-term combined intake need to be considered. Therefore,
it is important to carefully evaluate the safety of foodadditives.
It has been reported that artificial sweeteners causechanges in the intestinal flora, dysbiosis, thus inducing_ 14 0 _The authors claim no conflict of interest in this study.

Glycative Stress Research

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