Effect of chronic exposure to aspartame on oxidative stress in the

ASHOK IYYASWAMY and SHEELADEVI RATHINASAMY*

Department of Physiology, Dr. ALM PG Institute of Basic Medical Sciences,
University of Madras, Sekkizhar campus, Taramani, Chennai 600 113, India
*Corresponding author (Fax, +91-44-24540709; Email, drsheeladevi@unom.ac.in)
This study was aimed at investigating the chronic effect of the artificial sweetener aspartame on oxidative stress in
brain regions of Wistar strain albino rats. Many controversial reports are available on the use of aspartame as it
releases methanol as one of its metabolite during metabolism. The present study proposed to investigate whether
chronic aspartame (75 mg/kg) administration could release methanol and induce oxidative stress in the rat brain. To
mimic the human methanol metabolism, methotrexate (MTX)-treated rats were included to study the aspartame
effects. Wistar strain male albino rats were administered with aspartame orally and studied along with controls and
MTX-treated controls. The blood methanol level was estimated, the animal was sacrificed and the free radical changes
were observed in brain discrete regions by assessing the scavenging enzymes, reduced glutathione, lipid peroxidation
(LPO) and protein thiol levels. It was observed that there was a significant increase in LPO levels, superoxide
dismutase (SOD) activity, GPx levels and CAT activity with a significant decrease in GSH and protein thiol.
Moreover, the increases in some of these enzymes were region specific. Chronic exposure of aspartame resulted in
detectable methanol in blood. Methanol per se and its metabolites may be responsible for the generation of oxidative
stress in brain regions.
[Iyyaswamy A and Rathinasamy S 2012 Effect of chronic exposure to aspartame on oxidative stress in the brain of albino rats. J. Biosci.
37 679688] DOI 10.1007/s12038-012-9236-0
1. Introduction
Aspartame (L-aspartyl-L-phenylalanine methyl ester), a low-
calorie sweetener, is about 180 times sweeter than normal
sugar and is used in over 5000 foods and beverages in
todays market. The use of aspartame by diabetic individuals
is increasing; it is widely used in the weight loss regime, and
approximately 200 million people consume aspartame
worldwide (Shapiro 1988). In 1965 aspartame was discov-
ered by James Schlatter of the G.D. Searle Company
(Garriga and Metcalfe 1988). Aspartame has a biological
effect even at the recommended daily dose (Gombos et al.
2007). Approximately 50% of the aspartame molecule is
phenylalanine, 40% is aspartic acid and 10% is methanol
(Newsome 1986). Aspartic acid is a metabolite of aspartame
that is an excitatory amino acid and is normally found in
high levels in the brain. These levels are controlled by the
bloodbrain barrier, which protects the brain from large
fluctuations in plasma aspartate (Maher and Wurtman
1987). Phenylalanine is an amino acid essential to the pro-
duction of monoamines in the brain and is found in nearly all
protein foods (Fernstrom et al. 1983). On the other hand, due
to the high levels of phenylalanine in blood, consumption of
aspartame may cause brain damage (Haschemeyer and
Haschemeyer 1973). Certain brain amino acid levels have
been shown to be increased after aspartame consumption
(Dailey et al. 1991; Diomede et al. 1991; Yokogoshi et al.
1984). Although methanol is released during aspartame di-
gestion, it only accounts for 10% of the aspartame molecule
(Stegink et al. 1983), and among the metabolites, methanol
is a toxicant that causes systemic toxicity (Kruse 1992).
Various neurochemical effects due to aspartame con-
sumption have been reported (Coulombe and Sharma 1986;
Pan-Hou et al. 1990; Goerss et al. 2000). Their adverse
http://www.ias.ac.in/jbiosci J. Biosci. 37(4), September 2012, 679688, * Indian Academy of Sciences 679
Keywords. Aspartame; blood methanol; oxidative stress; rat folate-deficient model; free radical
Published online: 3 August 2012
brain of albino rats
neurological effects include headache (Johns 1986), insom-
nia and seizures after ingestion of aspartame, which are also
accompanied by the alterations in regional concentrations of
catecholamine (Coulombe and Sharma 1986). Previous find-
ing have shown that aspartate may lead to neurotoxicity
through sustained contact with the receptors, such as gluta-
mate producing an excitotoxic effect (Olney et al. 1980).
Large doses of both aspartame as well as these individual
metabolites have been tested in humans and other animals,
with controversial reports. It has been reported that not only
the metabolites of methanol, but methanol per se as well, is
toxic to the brain (Jeganathan and Namasivayam 1998). The
primary metabolic fate of methanol is the direct oxidation to
formaldehyde and then into formate. The toxic effects of
methanol in humans are due to the accumulation of its
metabolite formate (Tephly and McMartin 1984; Tephly
1991). The severity of clinical findings in methanol intoxi-
cation correlated better with formate levels (Osterloh et al.
1986). Formate is metabolized twice as fast in rat as in
monkey (McMartin et al. 1978). The metabolism of formate
is mediated through a tetra-hydro-folate-dependent pathway
(Eells et al. 1982). Humans and non-human primates are
uniquely sensitive to methanol poisoning because of their
low liver folate content (Johlin et al. 1987). There are pro-
found differences in the rate of formate oxidation in different
species which determine their sensitivity to methanol
(Mcmartin et al. 1978; Eells et al. 1983). In non-human
primates and humans, alcohol dehydrogenase mediates this
reaction (Makar et al. 1990). In rats and other non-primate
species, this reaction is mediated by catalase. Microsomal
oxidizing system is reported to be responsible for free radical
generation. In addition to that, inhibition of cytochrome
oxidase by formate leads to the generation of superoxide,
peroxyl and hydroxyl radicals. Tephly (1991) also proved
that a catalase system is also one of the major pathways of
methanol oxidation in rat hepatocytes. Free methanol is
created from aspartame when it is heated to above 86F
(30C). This would occur when an aspartame-containing prod-
uct is improperly stored or when it is heated (e.g. as part of a
food product such as Jello). Methanol is gradually released in
the small intestine when the methyl group of aspartame
encounters the enzyme chymotrypsin. Methanol breaks down
into formic acid and formaldehyde in the body. Formaldehyde
is a deadly neurotoxin (Lee et al. 1994; Eells et al. 2000).
Rodents do not develop metabolic acidosis during methanol
poisoning, owing to their high liver folate content, and in
order to create similar results in human beings, only folate-
deficient rodents are required to accumulate formate in order
to develop acidosis in methanol poisoning (Lee et al. 1994;
Eells et al. 2000).
Hence, in this study, in order to mimic the human
situation, a folate deficiency status was induced by ad-
ministering methotrexate (MTX). The focus of this study
is to investigate whether the chronic oral administration
of aspartame (75 mg/kg) can release methanol as a by-product
after metabolism and whether it induces oxidative stress in the
rat brain regions after aspartame administration.
2. Materials and methods
2.1 Animals
Wistar strain male albino rats (200220 gm) were main-
tained under standard laboratory conditions with water and
food. For the folate-deficient group, folate-deficient diet was
provided for 45 days prior to the experiment and MTX was
administered for a week before the experiment. The animals
were handled according to the principles of laboratory care
framed by the committee for the purpose of Control and
Supervision of Experiments on Animals (CPCSEA),
Government of India. Prior to the experimentation, proper
approval was obtained from the Institutional Animal Ethical
Committee (No: 01/032/2010).
2.2 Chemicals
Aspartame, methotrexate and malondialdehyde were pur-
chased from Sigma-Aldrich.Co., St. Louis, USA. All the
other chemicals were of analytical grade obtained from
Sisco research laboratory, Mumbai, India.
2.3 Experimental design
The rats were divided in to three groups, namely, saline
control, MTX-treated control, and MTX-treated aspartame-
administered groups. Each group consisted of six animals.
Aspartame mixed in sterile saline was administered orally
(75 mg/kg body weight), based on the earlier report (Leon
et al. 1989). However, early reports on aspartame of higher
doses included two different authors, namely, Labra-Ruiz et
al. (2007) and Leon et al. (1989), and the reports are con-
troversial. This provided additional interest to use this dos-
age in our study. In order to confine within the human
exposure limit, this dose was selected. A 1 L (approx. 1
quart) aspartame-sweetened beverage contains about 56 mg
of methanol was used. Heavy users of aspartame-containing
products consume as much as 250 mg of methanol daily, or
32 times the EPA limit.
MTX in sterile saline was administered (0.2 mg/kg/day)
subcutaneously for 7 days to folate-deficient treated as well
as to folate-treated aspartame groups (Gonzalez-Quevedo
et al. 2002). One week after treatment with MTX, folate
deficiency was confirmed by estimating the urinary excretion
of formaminoglutamic acid (FIGLU) (Tabor and Wyngarden
1962). From the eighth day, only the MTX-treated aspartame
680 Ashok Iyyaswamy and Sheeladevi Rathinasamy
J. Biosci. 37(4), September 2012
group received the aspartame, whereas the other two groups
received equivalent volumes of saline as an oral dose and all
animals were handled similarly. The chronic dose of aspar-
tame was given for 90 days and all the animals were fed
folate-deficient diet except the control animals till 90 days.
2.4 Sample collections
The blood samples and isolation of brain was performed
between 8 and 10 a.m. to avoid circadian rhythm induced
changes. Stress-free blood samples were collected as per the
technique described by Feldman and Conforti (1980).
2.5 Brain dissection
The animals were sacrificed and the brain was immediately
removed and washed with ice-cold phosphate buffered saline
(PBS). To expose the brain, the tip of curved scissors was
inserted into the foramen magnum and a single lateral cut
was made into the skull extending forward on the left and
right side. With a bone cutter, the dorsal portion of cranium
was peeled off, and using a blunt forceps, the brain was
dropped onto the ice-cold glass plate, leaving the olfactory
bulbs behind. The whole process of removing brain took less
than 2 min. After removing the brain, it was blotted and
chilled. Further dissection was made on ice-cold glass plate.
The discrete regions of brain (cerebral cortex, cerebellum,
midbrain, pons medulla, hippocampus and hypothalamus)
were dissected according the method given by Glowinski
and Iverson (1996). The homogenate (10%w/v) of the indi-
vidual regions were prepared in a Teflon-glass tissue ho-
mogenizer, using ice-cold Tris HCl (100 mm, pH 7.4) buffer
and centrifuged separately in refrigerated centrifuge at 300g
for 15 min. The supernatant was used for analysing the
parameters in this study.
2.6 Blood methanol measurement
100 mL plasma was deproteinized with equal volume of
acetonitrile and centrifuged for 7 min at 4C (Dorman et al.
1994). The supernatant (20 mL) was analysed for blood
methanol and formate using a HPLC refractive index detec-
tor system (Shimadzu RID, Japan) (equipped with Rezex
ROA-organic acid column 300 mm7.5 mm I.D.,
Phenomenex) with the security guard cartridge (AJO 4490
Phenomenex). Column oven was used to maintain the tem-
perature at 60C. The mobile phase was 0.026 N sulphuric
acid (Pecina et al. 1984). By using methanol as an external
standard, the recovery of methanol (HPLC grade) from
blood was found to be 9296%. Linearity for methanol
was found to be 5500 mg/100 mL. The detector sensitivity
for methanol was found to be 5 mg/100 mL and reproduc-
ibility was>93%.
2.7 Plasma corticosterone
Plasma corticosterone level was estimated according to the
method described earlier by (Mattingly 1962) using excita-
tion at 470 nm and emission at 530 nm in fluorescence
spectrophotometer.
2.8 Lipid peroxidation
15 gm trichloro acetic acid was added to 100 mL of 0.25 N
hydrochloric acid. 15 mg of thio barbituric acid was dissolved
in 4 mL of trichloro acetic acid in HCl mixture. To 0.1 mL of
homogenate, 0.4 mL trichloro acetic acidthio barbituric acid
hydrochloric acid mixture was added and kept in a boiling
water bath for 20 min. Then, it was cooled to roomtemperature
gradually, followed by addition of 1 mL of n-butanol, vor-
texed well and centrifuged for 10 min. The supernatant was
taken and was read at 532 nm in spectrophotometer. The
level of LPO was expressed as nanomoles of malondialde-
hyde (an intermediary product of lipid peroxidation, using
thio barbituric acid)/mg protein (Ohkawa et al. 1970).
2.9 Superoxide dismutase (EC 1.15.1.1) (SOD)
0.5 mL of homogenate was mixed with 0.5 mL of ethanol
chloroform mixture; the content was shaken well for 15 min
and the centrifuged for 10 min. To 0.5 mL of supernatant
taken, 2 mL of Tris-HCl buffer (pH 8.2) was added. Finally,
0.5 mL of freshly prepared pyrogallol solution was added
and read at 470 nm in 0, 1, 2 and 3 min intervals. The
activity was expressed as Units/min/mg protein (Marklund
and Marklund 1974).
2.10 Catalase (EC.1.11.1.6)(CAT)
Four sets of 0.1 mL of homogenate was mixed with 1 mL of
phosphate buffer and 0.5 mL of H
2
O
2
, and this reaction was
arrested by addition of 2 mL potassium dichromate acetic
acid reagent at various intervals of 0, 15, 30 and 60 s. Then
all the samples were kept in a boiling water bath for 10 min
and were cooled gradually. The developed green colour was
read at 610 nm. The activity was expressed amount of H
2
O
2
utilized/min/mg protein (Sinha 1972).
2.11 Glutathione peroxidase (EC.1.11.1.9) (GPx)
To three sets of test tubes 0.4 mL of phosphate buffer,
0.1 mL of sodium azide, 0.2 mL of standard GSH solution
Effect of aspartame on oxidative stress 681
J. Biosci. 37(4), September 2012
and 1 mL of H
2
O were sequentially mixed. To this 0.1 mL of
H
2
O
2
was added and the reaction in individual set was
arrested with 1 mL of 10% TCA at 0, 1.5, 3 min intervals
and then centrifuged for 10 min. To the 0.2 mL of superna-
tant 1.8 mL of H
2
O followed by 1 mL of 5% TCA were
added and allowed to stand at room temperature for 20 min.
Then 4 mL of phosphate solution was added, followed by
0.5 mL of DTNB. The optical density was taken at 412 nm
within 5 min. The GPx activity is expressed as units/min/mg
protein (Rotruck et al. 1973).
2.12 Reduced glutathione
To 0.2 mL of homogenate, 1.8 mL of H2O, 1 mL of 5%
TCA were sequentially added and kept at room temperature
for 20 min. Then, 4 mL of 0.3 M phosphate solution fol-
lowed by 0.5 mL of DTNB was added and mixed well. The
optical density was taken at 412 nm within 5 min. from the
addition of DTNB. Reduced glutathione (GSH) was mea-
sured by its reaction with 5.5- dithiobis 2 nitro benzoic acid
(DTNB), to form a compound that absorbs at 412 nm
(Moron et al. 1979). The level of GSH is expressed as g
of GSH/mg of protein.
2.13 Protein thiol
To 1 mL of homogenate was added 1.5 mL of Tris-
hydrochloric acid buffer (pH 8.2) which contained 0.02
ethylene di-amine tetra acetic acid . This was followed by
the addition of 0.1 mL of DTNB solution and 7.5 mL meth-
anol. The contents were mixed well in a vortex mixture and
than centrifuged at 3000g for 10 min. The colour developed
was read at 412 nm. The level of thiol was expressed as
g/mg protein. Tissues were analysed for protein and
sulfhydryl concentration (Sedlack and Lindsay 1968).
2.14 Statistical analysis
Statistical analysis was carried out using the SPSS statistical
package version 17.0. The results are expressed as mean STD
and the data were analysed by the one-way analysis of variance
(ANOVA) followed by Turkeys multiple comparison tests
when there was a significant F test ratio. The level of signif-
icance was fixed at p0.05.
3. Results
The data from various groups for the individual parameters
are presented as bar diagram with mean STD.
3.1 Blood methanol level and plasma corticosterone level
The data for the blood methanol level and plasma cortico-
sterone level after chronic exposure to aspartame adminis-
tration are given in figure 1. Even after chronic exposure, the
aspartame-treated MTX animals showed marked increase in
the blood methanol level (df 2, F=2789) and plasma corti-
costerone level (df 2, F=874.6) from controls as well as from
the MTX-treated group.
3.2 Lipid peroxidation, superoxide dismutase
and catalase levels
The results are given in figure 2. The LPO levels in the
lipid peroxidation (LPO) MTX-treated animals did not differ
from those in the controls. However, the aspartame-treated
MTX animals showed a marked increase in the LPO level in
the entire brain regions studied, which included cerebral
cortex (df 2, F=182), cerebellum (df 2, F=125.8), midbrain
(df 2, F=272.2), pons-medulla (df 2,F=206.5), hippocampus
(df 2, F=187.4) and hypothalamus (df 2, F=235.7) from the
control as well as from the MTX-treated animals.
The results are given in figure 3. The superoxide dismu-
tase (SOD) activity in the MTX-treated animals did not
differ from those in the controls. However, the aspartame-
treated MTX animals showed a marked increase in the SOD
activity as compared to control and MTX-treated animals, in
the cerebral cortex (df 2, F=50.5), cerebellum (df 2, F=
19.2), midbrain (df 2, F=46.5), pons-medulla (df 2, F=
90.3) hippocampus (df 2, F=106) and hypothalamus(df 2,
F=84.8) from. The results are given in figure 4. The CAT
activity in the MTX-treated animals did not differ from the
controls. However, the aspartame-treated MTX animals
showed a marked increase in the CAT activity as compared
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Figure 1. Methanol level (mM) in rat blood upon aspartame administration (75 mg/kg) using HPLC and plasma corticosterone level.
682 Ashok Iyyaswamy and Sheeladevi Rathinasamy
J. Biosci. 37(4), September 2012
to control and MTX treated animals, in the cerebral cortex
(df 2, F=130.2), pons-medulla (df 2, F=51), hippocampus
(df 2, F=169) and hypothalamus (df 2, F=289.8). In the
midbrain (df 2, F=196.7) and cerebellum (df 2, F =126),
only a marked increase in CAT activity as compared to
control was seen and this remained similar to MTX-treated
animals.
3.3 Glutathione peroxidase, reduced glutathione
and protein thiol levels
The results are given in figure 5. The GPx activity in the
MTX-treated animals did not differ from the controls.
However, the aspartame-treated MTX animals showed a
marked increase in the GPx activity as compared to
control and MTX-treated animals, in tge cerebral cortex
(df 2, F=35.4) and midbrain (df 2, F=89.9). In the cerebellum
(df 2, F=83.6), pons-medulla (df 2, F=67.7), hippocampus (df
2, F=81.7) and hypothalamus (df 2, F =136.9), a marked
increase in GPx activity as compared control was seen, and
this remained similar to MTX-treated animals. The results are
given in figure 6.The GSH levels in the MTX-treated animals
did not differ from the controls. However, the aspartame-
treated MTX animals showed a marked decrease in the GSH
levels as compared to control and MTX-treated animals, in
the cerebral cortex (df 2, F =21.4), midbrain (df 2, F=28.7),
pons-medulla (df 2, F=23.1) and hippocampus (df 2, F=
14.62). However, in the hypothalamus (df 2, F=19.4) the
GSH level showed marked decrease as compared to controls
but not the MTX-treated group. Moreover, in the cerebellum,
the GSH level remained similar to that in control and MTX-
treated animals. The results are given in figure 7. The protein
thiol levels in the MTX-treated animals did not differ from
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LPO activity
Figure 2. Effect of aspartame (75 mg/kg body weight) on LPO activity (mmol/mg tissue) in rat brain discrete regions.
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SOD Activity
Figure 3. Effect of aspartame (75 mg/kg body weight) on SOD activity (units/mg tissue) in rat brain discrete regions.
Effect of aspartame on oxidative stress 683
J. Biosci. 37(4), September 2012
those in the controls. However, the aspartame-treated MTX
animals showed a marked decrease in the protein thiol levels
as compared to control and MTX-treated animals, in the
entire brain regions studied such as the cerebral cortex (df
2, F=114.7), cerebellum (df 2, F=158.9), midbrain (df 2, F=
83.7), pons-medulla (df 2, F=37.1), hippocampus (df 2, F=
68.6) and hypothalamus (df 2, F=119.3).
4. Discussion
This study deals with strengthening the toxic metabolite
methanol released in the body after the consumption of
aspartame. The alteration in the free-radical-scavenging
enzymes in the aspartame-administered animals clearly indi-
cates that free radical generation may be due to the methanol
which is one of the metabolic products of aspartame. Even
after chronic aspartame administration, there was detectable
blood methanol in the aspartame-treated animals. Methanol
is metabolized by three enzyme systems, namely, the alcohol
dehydrogenase system, the catalase peroxidative pathway
and the microsomal oxidizing systems. Among these, the
microsomal oxidizing system is reported to be responsible
for free radical generation (Goodman and Tephly 1968).
Exposure to methanol causes oxidative stress by altering
the oxidant/antioxidant balance in lymphoid organs of rat
(Parthasarathy et al. 2006). The condition could also be
associated with the abundance of redox active transition
metal ions, and the relative death of antioxidant defence
system (Samuel et al. 2005). In addition to that, oxidative
stress may lead to the generation of superoxide, peroxyl and
hydroxyl radicals (Keyhani and Keyhani 1980).The increase
in free radicals could not be ignored as cells can be injured or
killed when the ROS generation overwhelms the cellular
ant i oxi dant capaci t y (Oberl y and Oberl y 1986).
Particularly, the brain is more vulnerable to oxidative
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CAT activity
Figure 4. Effect of aspartame (75 mg/kg body weight) catalase activity (units/mg tissue) in rat brain discrete regions.
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GPx Activity
Figure 5. Effect of aspartame (75 mg/kg body weight) on GPx activity (units/mg tissue) in rat brain discrete regions.
684 Ashok Iyyaswamy and Sheeladevi Rathinasamy
J. Biosci. 37(4), September 2012
damage due its high oxygen consumption and due to the
presence of high levels of polyunsaturated fatty acids (Floyd
and Carney 1992).
SOD converts superoxide anion (O
2

) to hydrogen per-
oxide (H
2
O
2
) (Akyol et al. 2002), which are subsequently
converted to water and molecular oxygen by glutathione
peroxidase or catalase (Dringen 2000). GPx is a major anti-
oxidant enzyme in many tissues and has been speculated to
be a major antioxidative mechanism in the brain (Barlow-
Walden et al. 1995). In this study there was a significant
increase in the SOD activity after chronic aspartame inges-
tion with an associated increase in the catalase activity. As
catalase is the main scavenger of H
2
O
2
at high concentration
(Kono and Fridorich 1982), that indicates the accumulation
of H
2
O
2
as a result of increased SOD activity. It has been
found that consumption of methanol provokes changes in the
activity of antioxidant enzymes with an increase in the
activity of catalase (Skrzydlewska et al. 1998). Hence, the
increase in this enzyme activity may also be due to the free
radicals generated. According to Yu (1994), free radicals
may also induce the expression of antioxidant enzymes,
thereby enhancing the neuronal resistance to subsequent
oxidative challenges.
LPO is a free-radical-mediated process. In the entire rat
brain regions after aspartame consumption, a marked in-
crease in LPO was noted, which also supports the generation
of free radicals. Generally, when the generation of reactive
free radicals overwhelms the antioxidant defense, LPO of the
cell membrane occurs. In spite of the increase in the SOD,
CAT and GPx enzyme system in order to prevent the accu-
mulation of free radical, a marked increase in the LPO in the
entire brain regions indicated this result and possible loss of
membrane integrity. Moreover, reactive-oxygen-mediated
damage could not be overlooked particularly to proteins,
which has been shown to deleteriously affect cellular func-
tions (Sohal et al. 2002).
GSH is considered to be one of the most important com-
ponents of the antioxidant defense of living cells. The re-
duced tri-peptide GSH is a hydroxyl radical and singlet
oxygen scavenger, and participates in a wide range of cellu-
lar functions. Since GSH is present in high intracellular
concentrations, there is a high probability that reactive
oxygen species (such as superoxide, singlet oxygen and
hydrogen peroxide) is to be quenched by reaction with
GSH before they can initiate their chain reaction-
damaging effects (Jones et al. 1981). The depletion of
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Reduced GSH Level
Figure 6. Effect of aspartame (75 mg/kg body weight) on GSH concentration (mmol/mg tissue) in rat brain discrete regions.
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Protein Thiol Level
Figure 7. Effect of aspartame (75 mg/kg body weight) on protein thiol levels (mmol/mg tissue) in rat brain discrete regions. CC, cerebral cortex;
CB, cerebellum; MB, mid brain; PM, pons medulla; HI, hippocampus; HY, hypothalamus. The level of significance was fixed at p0.05 (n= 6).
Effect of aspartame on oxidative stress 685
J. Biosci. 37(4), September 2012
GSH may be one of the reasons for the increase in the
vulnerability added to free-radical-induced damage. In addi-
tion, the decrease in GSH concentration is quite possible
because the methanol metabolism also depends upon GSH
(Pankow and Jagielki 1993).
The significant decrease in the protein thiols observed in
this study may be due to the oxidation of proteins in oxida-
tive process, and it is also justified by the decreased level of
one of the major thiol substance GSH. Similarly decreased
protein thiols in brain due to oxidative damage was reported
by Patsoukis et al. (2004), and Nikolaos et al. (2004) support
the present findings.
From the foregoing it is clear that the methanol,
which is a by-product of aspartame, may be responsible
for the alteration observed in the free-radical-scavenging
system. Since methanol is freely permeable through
membranes and lipids, it also gets distributed in the
brain tissues and may cause the damage. Increased pro-
duction of free radicals and increased oxidative damage
to proteins in distinct brain regions, retina and optic nerve
after methanol administration (Rajamani et al. 2006) was also
reported earlier lending support to the present findings. The
scientific reports on aspartame also revealed that aspartame
consumption affects the brain. The aspartame intake has
been reported to be responsible for neurological and behav-
ioural disturbances in sensitive individuals (Johns 1986).
Moreover, Mourad and Noor (2011) have observed that the
daily ingestion of aspartame for 4 weeks induces significant
increase in the LPO levels in the cerebral cortex, which is
accompanied by significant decrease in GSH content and a
significant increase in SOD activity, which is also in agree-
ment with this study. According to them, the decrease in
GSH content and increase in SOD activity persisted until
after 6 weeks of aspartame treatment. Moreover, they add
that aspartame-induced oxidative stress may depend on the
duration of aspartame administration even within the accept-
able daily intake dose. The present study reveals that aspar-
tame administration in the body system persists for longer
duration, which indicates the possible accumulation of meth-
anol and its metabolite. Since it is consumed more by dia-
betic people whose metabolism is already altered, it is
essential to do more work on these lines and create aware-
ness regarding the usage of this artificial sweetener.
5. Conclusion
After chronic exposure of aspartame, detectable methanol con-
tinues to circulate in the blood; methanol per se and its metab-
olites may be responsible for the generation of oxidative stress in
brain regions. This study confirms the presence of toxic metab-
olite after aspartame administration and it emphasises the need
to caution the people who are using aspartame routinely.
Acknowledgements
The author is grateful for the valuable suggestion offered by
Dr NJ Parthasarathy and Dapkupar Wankhar. The financial
assistance provided by the University of Madras is gratefully
acknowledged.
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MS received 18 April 2012; accepted 06 June 2012
Corresponding editor: INDRANEEL MITTRA
688 Ashok Iyyaswamy and Sheeladevi Rathinasamy
J. Biosci. 37(4), September 2012