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ON THE POSSIBLE INVOLVEMENT OF AN OPIOIDERGIC MECHANISM IN THE ANTINOCICEPTIVE EFFECT OF ASPARTAME IN MICE (Mus musculus)

Maria Luisa J. AgravanteFaye B. Garciano

Submitted to theDepartment of Biology

College of Arts and SciencesUniversity of the Philippines Manila

Padre Faura, Manila

In partial fulfillment of the requirementsFor the degree of

Bachelor of Science BiologyMarch 2008

Antinociceptive Effect of AspartameAgravante and Garciano, 2008

ii

Department Of BiologyCollege of Arts and Sciences

University of the Philippines – ManilaPadre Faura, Manila

Announcement ofUndergraduate Thesis Presentation

MARIA LUISA JOSE AGRAVANTEFAYE BONDOC GARCIANO

Entitled

ON THE POSSIBLE INVOLVEMENT OF AN OPIOIDERGIC MECHANISM IN THE ANTINOCICEPTIVE EFFECT OF ASPARTAME IN MICE (Mus musculus)

For the degree ofBachelor of Science in Biology

2:00 PM, 10 March 2008Room 11C, Rizal Hall

THESIS ADVISER THESIS CO-ADVISERMaria Ofelia M. Cuevas, M.S. Miriam P. de Vera, M.S.Associate Professor Assistant ProfessorDepartment of Biology Department of BiologyUP Manila UP Manila

THESIS READER THESIS READERElisa L. Co, Ph.D. Kimberly S. Beltran, M.S.Associate Professor InstructorDepartment of Biology Department of BiologyUP Manila UP Manila

Endorsed by: Authorized by:

Maria Ofelia M. Cuevas, M.S. Arnold V. Hallare, Ph.D.Chair ChairThesis Committee Department of BiologyCAS, UP Manila


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Department of BiologyCollege of Arts and Sciences

University of the Philippines – ManilaPadre Faura, Manila

ENDORSEMENT

The thesis attached hereto, entitled “On the Possible Involvement of an Opioidergic Mechanism in the Antinociceptive Effect of Aspartame in Mice” prepared and submitted by Maria Luisa Jose Agravante and Faye Bondoc Garciano, in partial fulfilment of the requirements for the degree of Bachelor of Science in Biology was successfully defended on March 10, 2008.

MARIA OFELIA M. CUEVAS, M.S. MIRIAM P. DE VERA, M.S. Thesis Adviser Thesis Co-Adviser

ELISA L. CO, Ph.D. KIMBERLY S. BELTRAN, M.S. Thesis Reader Thesis Reader

This undergraduate thesis is hereby officially accepted as partial fulfilment of the requirements for the degree of Bachelor of Science in Biology.

ARNOLD V. HALLARE, Ph.D. REYNALDO H. IMPERIAL, Ph.D.Chair DeanDepartment of Biology College of Arts and Sciences


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BIOGRAPHICAL DATA

I. Personal Information

Name: Maria Luisa Jose AgravanteNickname: RiaBirthday: March 20, 1986Birthplace: Manila, PhilippinesAddress: 25 St. Joseph Street, Paradise Village

Project 8, Quezon CityMobile number: 09189918319Email address: [email protected]: Julian Federico Agravante

Luz Mary Lou Jose Agravante

II. Educational Background

Primary: Colegio San Agustin (1993-2000)Dasmariñas Village, Makati City, Philippines

Secondary: Colegio San Agustin (2000-2004)Dasmariñas Village, Makati City, Philippines

Collegiate: University of the Philippines – Manila (2004-2008)Padre Faura Street, Ermita, Manila

III. Organizations

Member, Biological Sciences SocietyMember, Biology Majors’ AssociationMember, Quod Erat DemonstrandumMember, UP Forensic Society


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BIOGRAPHICAL DATA

I. Personal Information

Name: Faye Bondoc GarcianoNickname: FayeBirthday: March 19, 1988Birthplace: Quezon City, PhilippinesAddress: 30 Libra Street Camella Homes II-B

Bacoor, CaviteMobile number: 09172414017Email address: [email protected]: Filomeno Rebano Garciano, Jr.

Corcini Bondoc Garciano

II. Educational Background

Primary: Southville International School (1994-1998, 1999-2000)B.F. Homes International, Las Piñas City

Philippine School Doha (1998-1999)Doha, Qatar

Secondary: Statefields School, Inc. (2000-2004)National Road, Molino III, Bacoor, Cavite

Collegiate: University of the Philippines Manila (2004-2008)Padre Faura Street, Ermita, Manila

III. Organizations

Vice President, Biology Majors’ AssociationMember, Biological Sciences SocietyMember, Quod Erat Demonstrandum


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ACKNOWLEDGEMENT

(Sgd.) (Sgd.) Maria Luisa J. Agravante Faye B. Garciano


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TABLE OF CONTENTS

TITLE PAGE ............................................................................................................. i

ANNOUNCEMENT .................................................................................................. ii

ENDORSEMENT ...................................................................................................... iii

BIOGRAPHICAL DATA .......................................................................................... iv

ACKNOWLEDGEMENT ......................................................................................... vi

TABLE OF CONTENTS ........................................................................................... vii

LIST OF TABLES..................................................................................................... viii

LIST OF FIGURES ................................................................................................... ix

LIST OF APPENDICES........................................................................................... . x

ABSTRACT ............................................................................................................... xi

INTRODUCTION..................................................................................................... 1

REVIEW OF RELATED LITERATURE ................................................................ 5

MATERIALS AND METHODS............................................................................. 9

RESULTS................................................................................................................. 13

DISCUSSION .......................................................................................................... 15

CONCLUSION........................................................................................................ 19

RECOMMENDATIONS......................................................................................... 20

LITERATURE CITED ............................................................................................. 21

APPENDICES........................................................................................................... 27


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List of Tables

PAGE

Table 1. Mean weights of mice negative control groups and aspartame-treatedgroups.................................................................................................................. 25

Table 2. Mean amount of aspartame solution consumed by mice...................... 25

Table 3. Mean index of analgesia (±S.E.M) of mice negative control groups and aspartame-treated groups…………………..…............................................ 25

Table 4. Mean differences of mice negative control groups and aspartame-treated groups………………….…………..………………………………….... 26


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List of Figures

PAGE

Figure 1. Mean index of analgesia of mice negative control groups andaspartame-treated groups…………………………………………………………. 28

Figure 2. Mean index of analgesia of mice negative control groups and aspartame-treated groups comparing the effect of aspartame after one and 14 days of administration. ………………………………………………………... 28

Figure 3. Mean index of analgesia of mice aspartame-treated groups comparing the effect of naloxone after one and 14 days of administration of treatment. ………………………………………………………………………. 29

Figure 4. Mean index of analgesia of mice negative control groups comparing the effect of naloxone after one and 14 days of administration of treatment. ………………………………………………………………………. 29


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List of Appendices

PAGE

Appendix A1. Structure of Aspartame (N-L-aspartyl-L-phenylalanine-1-methyl ester)……………………………………………………………………...... 31

Appendix A2. Structure of Naloxone molecule…………………………………… 31

Appendix B. Table of analysis of variance for within subject effects…………….. 32

Appendix C. Mean values of index of analgesia (± S.E.M.) of mice treatmentgroups (for all trial runs)……………………………………………………………33


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ABSTRACT

To test whether aspartame-induced antinociception involves opioidergic mechanisms, male IRC mice received either distilled water or aspartame solution (0.16% w/v) and then subcutaneously injected with either saline or naloxone, an opioid receptor antagonist. Aspartame solution and distilled water were given for one day or 14 days. Latency responses, measured via the hot plate and the tail flick test methods, were normalized into an index of analgesia. Within-subject interaction effects were significant for the variables of aspartame treatment, behavioral test applied, duration of administration and presence of naloxone. The mean index of analgesia of aspartame-treated mice was higher by 93% compared to that of the negative controls following a long-term (14-day) duration of administration. In addition, the presence of naloxone was observed to inhibit aspartame-induced antinociception during the said period. On the other hand, following short-term (24-hour) duration of administration, the mean index of analgesia of water-treated mice given with naloxone was higher by 135% compared to those of the negative controls. The findings suggest that opioidergic mechanisms and both the spinal and supraspinal structures may be involved in aspartame-induced antinociception which could be influenced by the duration of aspartame treatment. However, the results do not negate the possible transient antinociceptive effect of naloxone in the absence of aspartame.


1

INTRODUCTION

Background of the study

In recent years, much research has been conducted on the pain-relieving properties of

various substances. Sweet palatable substances such as sucrose, fructose, glucose, and saccharin,

have been reported to produce antinociception, a decrease in the response of an animal to

injurious stimuli. It is claimed that the mechanism of the analgesic effects produced by the

mentioned sweet substances may be attributed to their sweet taste, which triggers the release of

endogenous opiates in the body (Pepino and Mennella, 2005).

Aspartame is a non-caloric artificial sweetener that is widely used in a variety of food and

beverages. Discovered accidentally by American drug researcher James Schlatter in 1965, it goes

by the chemical name N-L-aspartyl-L-phenylalanine-1-methyl ester and is available

commercially under such brand names as NutraSweet® and Equal®. It is primarily composed of

two amino acids, aspartic acid and phenylalanine, wherein a liter of aspartame-sweetened soft

drink is said to contain about 400 mg of the former (Yellowlees, 1983; Romanowski, 2002). In

its pure form, aspartame appears as a white, odorless, crystalline powder and is said to be unique

for it provides sweetness without the caloric content that other sweeteners possess (Romanowski,

2002).

Since aspartame was made available commercially, much controversy has surrounded its

consumption. Articles and researches claim that the two amino acids in aspartame, phenylalanine

and aspartic acid, can damage the brain and cause other neurotoxic effects. However, these two

components of aspartame will only have adverse effects when taken by individuals suffering

from phenylketonuria and at very high doses (100mg/kg) (Stegink, 1980; Stegink, 1987). With


2

this, aspartame has been approved by the United States Food and Drug Administration as a

tabletop sweetener and is still widely used as of today (Romanowski, 2002).

Despite the abovementioned concerns, it has been suggested that aspartame exhibited

analgesic properties (Sharma, et al., 2005). Abdollahi and his collaborators suggested that the

inactivation of N-methyl-D-aspartate (NMDA) receptors by aspartame causes the analgesic

effect of the said compound (Abdollahi, et al., 2003). However, very little research has tested the

involvement of the endogenous opioid system in aspartame-induced antinociception. If naloxone,

an opioid receptor antagonist inhibits the antinociceptive effect of aspartame, then aspartame

induces a decrease in pain response by interacting with the endogenous opioid system.

Statement of the problem

Does the administration of aspartame in mice induce spinally-mediated antinociception

involving opioidergic mechanism?

Research objectives

This study aims to investigate whether the administration of aspartame induces opioid

receptor-mediated antinociception in mice based on behavioral response tests. It specifically

intends (1) to test whether the oral ingestion of aspartame ad libitum would result in the increase

in latency responses of male IRC mice; (2) to measure and compare the latency responses of

mice subjected to tests involving spinal and supraspinal structures; (3) to assess whether a short-

term and a long-term period of aspartame administration would increase pain tolerance in mice;

and (4) to measure and compare the latency responses, normalized to an index of analgesia, of

mice administered with an opioid receptor antagonist and a negative control.


3

Significance of the study

Concerns have been addressed with regards to anesthetic agents such as the drug

meperidine, which are used in the treatment of pain, particularly post-operative pain. It is said

that the use of such anesthetic agents pose the possibility of incurring respiratory problems like

respiratory depression and apnea. This concept is of primary concern both in children and adults

(Ren, et. al., 1997). Thus, other treatments to pain such as sweet-taste analgesia are being looked

upon as alternatives. Moreover, aspartame is a promising alternative for sucrose in producing

sweet-taste analgesia particularly to patients who are at risk of obesity since aspartame is a non-

caloric sweetener (Romanowski, 2002). Aspartame analgesia may also be an alternative to

sucrose analgesia for patients with diabetes since aspartame does not affect blood sugar level

(Lean and Hankey, 2004). Moreover, the data that will be gathered in this experiment may shed

light on the modulatory effect of aspartame in neural paths and neurotransmitters involved in

pain nociception.

Scope and Limitations

The experimental design was based on a randomized complete block design involving

four factors: aspartame treatment, duration of administration of treatment, type of analgesic test,

and the presence or absence of an antagonist which is naloxone. Antinociceptive effects via

induction of pain tolerance in mice were evaluated through behavioral tests involving spinal and

supraspinal mechanisms without invasive clinical procedures. The said behavioral nociceptive

tests only used thermal noxious stimuli. In addition, this study tested the short-term and long-

term effects of aspartame administration within a two-week period per trial run. However, a

dose-dependent antinociceptive effect of aspartame was not conducted due to constraints in the


4

supply of opioid antagonist. The experiment utilized repeated measures. Moreover, this study

examined the involvement of opioidergic mechanism in aspartame-induced antinociception.

However, the type of opioid receptor that aspartame activates was not specified.


5

REVIEW OF RELATED LITERATURE

Aspartame or N-L-aspartyl-L-phenylalanine-1-methyl ester (Appendix A1) is primarily

synthesized from phenylalanine and aspartic acid or aspartate. These two amino acids have a

sweet isomer and a bitter isomer. It is then a must for the appropriate isomers of phenylalanine

and aspartic acid to be used in synthesizing aspartame for it to be able to bind to the sweet

receptors of the tongue. This idea then accounts for the sweet taste of aspartame (Ophardt, 2003;

Romanowski, 2002).

Several researches have shown that sweet solutions, such as saccharin, sucrose, and

aspartame induce analgesia or antinociception. Analgesia is the absence of the sense of pain

without loss of consciousness. Antinociception, on the other hand, is defined as a decrease in

response of the sensory systems of the body to harmful or painful stimuli. Saccharin, the oldest

known artificial sweetener that goes under the brand name Sweet ‘N Low® by Cumberland

Packing Corporation in Brooklyn, New York, USA, has been shown to increase pain tolerance of

rats subjected to the hot plate test, which is a behavioral nociceptive test (Segato, et al., 1997).

Several studies regarding the analgesic effects of sucrose have also been conducted. Segato and

his team (1997) showed that orally-administered sucrose produced an increase in pain tolerance

in male albino Wilstar rats subjected to the tail flick test. Moreover, it was found that oral

administration of sucrose provided an increase in tolerance to persistent pain and hyperalgesia in

infant rats (Ren, et al., 1997).

Studies on the analgesic properties of sucrose have also been conducted in humans.

Johnston and others (2002) showed that routine intake of sucrose increased pain tolerance in

infants less than 31 weeks of age. The said infants were able to tolerate pain caused by invasive


6

and non-invasive but uncomfortable medical procedures such as injections and endotracheal tube

suctioning, respectively. Similar findings were shown by Ramenghi and his collaborators (1999),

wherein human infants of about 32 to 36 weeks showed an increase in pain tolerance to heel

pricks when administered with sucrose.

Aspartame has also been found to have analgesic properties. According to Abdollahi and

his team (2001), aspartame is able to control pain-related behavior through a nitric oxide/cyclic

guanosine monophosphate (cGMP)/glutamate release cascade by inactivating NMDA (N-

methyl-D-aspartate) receptors. NMDA receptors are ionotropic receptors for glutamate whose

activation results in the opening of an ion channel that is nonselective to cations. In addition,

another study by Abdollahi and others (2003) suggested that since aspartate, one of the major

components of aspartame is an excitatory amino acid (EA) like glutamate, it can interact with

NMDA receptors and modulate pain sensation.

Pepino and Mennella (2005) suggested that the analgesic effect of the previously

mentioned sweetening agents is said to involve afferent signals from the sense organ (Pepino and

Mennella, 2005). Ramenghi and his collaborators (1999) provided evidence wherein the direct

gastric loading of sucrose yielded no analgesic effects to human infants as compared to orally

administered sucrose wherein analgesia was observed. Also, the release of endogenous opiates is

said to be triggered by the taste of sweet substances since naloxone, a competitive opioid

receptor antagonist was said to be able to inhibit the analgesic effects of sucrose in rats (Segato,

et al., 1997). Naloxone acts by binding to the opioid receptors with a greater affinity than the

agonists for a specific binding site. It blocks the receptor so that the agonists cannot bind and

activate it (Sauro and Greenberg, 2005). However, it is a non-specific opioid antagonist. This

means that it can bind to any of the three types of opioid receptor, namely μ, δ, and κ. The


7

studies that were mentioned used the cold pressor, heel prick, and tail flick tests to assess the

antinociceptive properties of sweetening agents (Pepino and Mennella, 2005).

Typical behavioral tests that can be used to measure antinociception include the warm

water immersion tail flick, hot plate, acetic acid-induced writhing, and formalin tests. The warm

water immersion tail flick and hot plate tests are used to measure, via thermal stimulus of

cutaneous and short origin, the antinociceptive effect of different substances involving spinal and

supraspinal mechanisms respectively (South and Smith, 1998). The acetic-acid-induced writhing

and formalin tests on the other hand, use chemical stimuli (Chapman and Loeser, 1989).

The acetic acid-induced writhing test is a reflexive model wherein acetic acid is injected

into the animal and writhing or dorsoflexion of the back, stretching of hind limbs, and abdominal

contraction is considered as the animal’s pain response. In this model, the stimulus is applied for

a longer period of time (approximately 60 minutes). However, ethical issues are raised with

regards to this antinociceptive test since the pain stimulus (acetic acid) is applied for a longer

period of time and renders the animal unable to escape from the pain induced (Chapman and

Loeser, 1989).

One counterpart of the abovementioned analgesic test is the formalin test. It is similar to

the type and duration of stimulus application in the acetic acid-induced writhing test. Like the

hot plate test, it is an organized behavioral measure of nociception. The procedure involves a

dilute solution of formalin which is injected subcutaneously into one of the hind paws of the

animal. To measure the response in mice, the time spent licking or biting the injected part of the

foot is recorded (Chapman and Loeser, 1989).

The tail flick test is a reflexive measure of nociception wherein the reflex removal of the

tail is considered as a response (Chapman and Loeser, 1989). The hot plate test, on the other


8

hand, is an organized behavioral measure of nociception, wherein behavior toward a painful

stimulus is considered. This technique is also said to be the complement of the tail flick test,

since the hot plate test uses a thermal noxious stimulus as does the tail flick test. However, this

method involves supraspinal structures instead of spinal (South and Smith, 1998).

Since the first two tests mentioned, which use chemical stimuli pose ethical concerns, the

tail flick and hot plate tests were chosen for this present study to test whether the short-term and

long-term administration of aspartame would result in the decrease in the latency responses of

male IRC mice. Lastly, since no previous report has suggested the involvement of endogenous

opioid mechanisms in aspartame-induced antinociception, this present study will look into the

inhibitory effect of naloxone on the antinociceptive effects of aspartame, as an increase in

latency response of mice will indicate the involvement of endogenous opioid receptors.


9

MATERIALS AND METHODS

Experimental Animals

Forty-eight male IRC mice with a mean weight of 18 (± 0.4) grams each, purchased from

the Bureau of Animal Industries (BAI), Diliman, Quezon City, served as the test subjects in this

study.

Each mouse was weighed before acclimatization, and once a week after the habituation

period. The mice were housed two to a cage with a 12:12 hour light/dark cycle, with food and

water available ad libitum. In the subsequent experiment proper, the treatment solutions were

placed in feeding bottles and replaced each week. The amount of treatment solution consumed

by the treatment animals was estimated per day by weighing each feeding bottle at the start of

the day. To account the consumption of each mouse, the amount of treatment solution consumed

was divided by two since the mice were distributed two to a cage with one feeding bottle per

cage.

Chemicals

A one-kilogram pack of aspartame (99% purity) was purchased from Baler Industrial

Corporation in Quezon City. Normal saline solution (0.9% NSS) from a local medical supplies

store in Bambang, Manila and naloxone solution (0.4 mg/mL) from San Juan De Dios Hospital

in Pasay City were also purchased for the experiment.


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Experiment Proper

Three batches of test animals were used in this study, wherein a batch pertains to a single

run of the experiment. A period of roughly one to two months was observed between each run.

1. Treatment Groups

A single experimental run involved 16 mice randomly distributed into four groups of four

mice each. These four groups were designated as follows: (1) WS, which received distilled water

ad libitum, and was subcutaneously injected with saline, (2) WN, which received distilled water

ad libitum, and was subcutaneously injected with naloxone, (3) AS, which received aspartame

solution (0.16% w/v) ad libitum and was given saline, (4) AN, which received aspartame

solution (0.16% w/v) ad libitum and was given naloxone, where W is water, A is aspartame, S is

saline, and N is naloxone. Each group was then subdivided into two wherein the latency response

of the first subgroup was measured via hot plate, while that of the second subgroup was

measured via tail flick methods.

All mice were acclimatized for one week under standard laboratory conditions prior to

experimentation. The mice were then subjected to a habituation period for three days. This was

done in order for the mice to be accustomed to the researcher and to either the hot plate or the tail

flick apparatus. The mice in the first subgroup were habituated by placing each mouse on top of

the hot plate apparatus for thirty seconds. On the other hand, the mice in the second subgroup

were habituated by placing each mouse inside the restraining plastic cylinder and immersing the

tip of its tail in water for seven seconds.

On the day after the habituation period, baseline latencies were measured for each mouse.

Distilled water in feeding bottles was placed in the cages of the WN and WS groups, while


11

aspartame dissolved in drinking water (aspartame solution) in feeding bottles was placed in the

cages of the AN and AS groups.

Each mouse was then subjected to two latency measurements: the first conducted after

one day of distilled water or aspartame solution consumption, and the second conducted after 14

days. Latency responses were measured by either the hot plate test or the tail flick method.

2. Hot Plate Test

The hot plate test was performed as conducted by South and Smith (1998). The first

subgroups of mice (eight per trial run) were subjected to this test. Ten minutes prior to hot plate

latency (HPL) measurement, the WN and AN groups were subcutaneously injected with

naloxone solution (1mg/kg) while the WS and AS groups were subcutaneously injected with

saline. Each mouse was then placed on the hot plate apparatus heated to a temperature of 40 ±

0.5 ºC, which was surrounded by a transparent enclosure. The timer was stopped automatically

and the mouse was removed once the first behavioral sign of nociception (i.e. licking a hind paw,

vocalization, escape response) was observed and the duration of latent response (in seconds) of

which was recorded. When no sign of nociception is observed within 30 seconds, the mouse was

removed from the hot plate apparatus to prevent paw tissue damage. The period of 30 seconds

was then considered to be its HPL. Each HPL measurement was normalized by an index of

analgesia (IA) using the formula:

where IA is the Index of Analgesia, HPLtest is the hot plate latency after treatment, HPLcontrol is the

average of three hot plate latencies before treatment. The value 30 refers to the maximum time

(in seconds) of exposure to the thermal stimulus.

3. Tail Flick Method


12

The tail flick method was performed as conducted by Tabarelli and his collaborators

(2003). The second subgroups of mice (eight per trial run) were subjected to this method. Ten

minutes before tail flick latency (TFL) measurement, the WN and AN groups were

subcutaneously injected with naloxone while the WS and AS groups were subcutaneously

injected with saline. Then, each mouse was placed inside a plastic tube and the last three

centimeters of its tail was submerged into a hot water bath with a temperature of 52 ± 0.5 ºC.

The timer was stopped and the tail of the mouse was removed from the water bath once its tail

flicked in response to the thermal stimulus, the duration of latent response (in seconds) of which

was recorded. When the mouse failed to flick its tail within seven seconds, the tail was removed

from the water bath to prevent skin damage and the period of seven seconds was considered to

be its TFL. Each TFL measurement was normalized by an index of analgesia (IA) using the

formula:

where IA is the Index of Analgesia, TFLtest is the tail flick latency after treatment, TFLcontrol is the

average of three tail flick latencies before treatment. The value 7 refers to the maximum time (in

seconds) of exposure to the thermal stimulus.

Statistical analysis

All values were expressed as mean ± standard error of mean (S.E.M.). The data gathered

were subjected to repeated measures analysis of variance (ANOVA). The level of statistical

significance was set at p ≤ 0.5 level. The software Statistical Package for the Social Sciences

(SPSS) version 15.0 for Windows Operating System was the computation package utilized in the

study.


13

RESULTS

Measured Weights and Consumption Rates

During the course of each experimental run, the weight of each experimental mouse was

measured. On the average, each mouse showed a 23% increase in weight from the first day to the

fifteenth day of the experiment proper, and a 51% increase in weight from the first day to the last

day of the experiment proper (Table 1). In addition, throughout the course of the experiment

proper, the AS group consumed an average of 14.85 ml aspartame solution while the AN group

consumed an average of 11.92 of the said solution (Table 2).

Interaction Effect

The hot plate and the tail flick tests were used to assess the antinociceptive effect of

aspartame whether it involves supraspinal or spinal structures. The Analysis of Variance for

within subject effects showed that the interaction of duration, treatment (with or without

aspartame), behavioral test, and naloxone administration was not significant. The interaction

effect of variables, namely duration, treatment, naloxone, and experimental run was also not

significant (Appendix B).

In the subsequent sections, the effect of the particular independent variables involved has

incorporated the effect of the type of behavioural test employed.

Effect of Treatment and Duration of Administration

Aspartame treated groups administered with saline and negative control groups were

compared to test whether aspartame induces antinociception in mice. Following a 24-hour (short-


14

term) administration of treatment, results showed that the mean index of analgesia of the

aspartame treated groups AS did not differ significantly with that of the negative control groups

WS. On the other hand, for the 14-day (long-term) administration the reverse effect was

observed wherein the mean index of analgesia of the aspartame treated group S was higher by

95% compared to the negative control group WS14 (Table 3, Figures 1 and 2).

In order to test whether the antinociceptive effect of aspartame depends on the duration of

administration, the effect of aspartame after 24-hour administration of treatment was compared

to its effect after 14-day administration. Unlike in the 24-hour administration of treatment,

aspartame-induced antinociception was observed after 14 days of treatment since the mean index

of analgesia of the AS group was higher than that of the WS group after 14 days of

administration of treatment, while the mean index of analgesia of the AS group did not differ

significantly from that of the WS group after one day of administration of treatment (Table 3,

Figure 1 and 2).

Effect of Naloxone

Naloxone, an opioid receptor antagonist was used to test whether the antinociceptive

effect of aspartame involves opioidergic mechanism. If naloxone inhibits the antinociceptive

effect of aspartame, then aspartame-induced antinociception involves opioidergic mechanism.

Results show that following a 14-day administration of aspartame, the mean index of analgesia

of the AS group was higher by 93% compared to the AN group, the mean difference of which

lies within a 95% confidence interval of 0.046 to 0.704 (Table 4, Figures 1 and 3). However,

when treatment was administered for only one day, the mean latency response of the WN group

was higher by 135% compared to the WS group (Table 3, Figures 1 and 4).


15

DISCUSSION

Spinal and supraspinal structures may be involved in pain response or nociception

(Chapman and Loeser, 1989). The mechanism of aspartame-induced antinociception may be

assessed through the use of two different types of behavioral nociceptive tests. In the tail flick

test, the pain response is said to be processed at the spinal level whereas in the hot plate test,

nociception is mediated by supraspinal structures. Since the interaction effect of duration,

treatment (with or without aspartame), behavioral test, and naloxone administration was not

significant, the antinociceptive effect of aspartame involves both supraspinal and spinal

structures. Moreover, since the interaction effect of duration, treatment, naloxone, and

experimental run was not significant, the effect of all treatment combinations is consistent in all

three runs. Thus, it can be inferred that the test subjects are homogenous and extraneous

variables are minimal.

Regardless of the type of behavioral nociceptive test applied, the 14-day administration

of 0.16% (w/v) aspartame solution produced an observable antinociception in mice implying that

spinal and supraspinal structures are involved in aspartame-induced antinociception as was

mentioned in the previous paragraph. In accordance with the data, there are findings that show

that sweetening agents such as aspartame boost morphine-induced antinociception (Abdollahi, et

al., 2003). However, no antinociceptive effect of aspartame was detected after a short period of

24-hour administration only. Since the 24-hour and 14-day treatment administration were used

to compare the short and long-term effects of aspartame on antinociception, this result signifies

that the antinociceptive effect of aspartame is dependent on the duration of administration, which

was also observed for sucrose (Segato, et al., 1997).


16

The compound naloxone, an opioid receptor antagonist, was observed to inhibit the

antinociceptive effect of aspartame since the mean index of analgesia of the AS14 group is

higher compared to the AN14 group. These findings suggest that following the long-term

administration of aspartame, the antinociceptive effect of the said substance may involve

opioidergic mechanisms. These results are supported by the report of Segato and his

collaborators (1997) who revealed that antinociception induced by sweetening agents may

involve endogenous opioids.

The taste buds of the tongue are the primary sense organs which perceive gustatory

sensation. The facial nerve (cranial nerve VII) innervates the anterior two-thirds of the tongue,

while the glossopharyngeal nerve (cranial nerve IX) innervates the posterior third. The vagus

nerve (cranial nerve X) on the other hand, innervates the other taste buds. Taste information such

as sweet taste is then carried from the three mentioned cranial nerves to the nucleus of the

solitary tract which is part of the brain stem. From the said structure, the information is relayed

to certain regions of the brain, including the thalamus, cerebral cortex, amygdala, and

hypothalamus (Weiner, et al., 2003).

The hypothalamus contains the endogenous opioid β-endorphin (Shigeru, et al., 2003).

Nikfar and his peers (1997) reported that the antinociceptive effect of sweet substances such as

saccharin and sucrose was accompanied by an increase in the levels of β-endorphins in the

hypothalamus. Moreover, Taddio et al. (2003) stated that the sweet taste of sucrose triggered the

release of the said endogenous opioid. After being released, endogenous opioids bind to their

receptors. When β-endorphins bind to opioid receptors located in presynaptic terminals, a

decrease in calcium ion influx would result. The action potential brought about by pain stimulus

would then be less generated, thereby producing antinociception. On the other hand, when β-


17

endorphins bind to opioid receptors found in the postsynaptic terminals, there will be a resultant

increase in potassium ion efflux. This phenomenon then leads to postsynaptic hyperpolarization,

thus yielding a decrease in action potential generation brought about by pain stimuli.

Antinociception will thus occur (Golan, 2007). Therefore, when β-endorphin binds to opioid

receptors, modulation of pain is achieved (Kruger, 2001). Furthermore, the receptors of β-

endorphin is said to be located both in the brain and spinal cord, thereby suggesting the

involvement of spinal and supraspinal structures (Law, et al., 1979; Ferrara and Li, 1980).

Antinociception was also observed in water-treated, naloxone-administered groups for

the 24-hour administration of treatment since the mean index of analgesia of the WN1 group is

higher than that of the WS1 group. This suggests that naloxone administered at 1 mg/kg

subcutaneously may produce an analgesic effect in mice in the absence of aspartame. Atamer-

Simsek and his team (2000) reported that naloxone administered intraperitoneally at 2 mg/kg

was able to produce an antinociceptive effect by itself. In addition, Tsuruoka and his team (1998)

and Walker and her collaborators (1994) observed that antinociception induced by naloxone

administered intraperitoneally at 5 µg/kg and 5 mg/kg may involve serotonergic mechanisms.

Yohimbine, pirenperone, and ritanserin, which are antagonists of serotonergic receptors, were

able to reverse naloxone-induced antinociception. However, methiothepin, a 5-HT1 serotonergic

receptor antagonist, and MDL 72222, a 5-HT3 serotonergic receptor antagonist, did not inhibit

the antinociceptive effect of naloxone. These results suggest that the binding of naloxone to

opioid receptors induce antinociception involving 5-HT2 receptors (Walker, et al., 1994).

The hypothesis is that, naloxone, in acting on opiate receptors, may release serotonin (5-

hydroxytryptamine or 5-HT), which is involved in the regulation and processing of nociception

(Diaz-Reval, et al., 2002). The periaqueductal grey area (PAG) is the key part in the descending


18

inhibitory pathways, which regulate antinociception, in the central nervous system. Information

from different brain regions is received by the PAG, which is considered as the entry in the

modulation of nociception, particulary in the dorsal horn. The nucleus raphe magnus (NRM) and

some fibers in the spinal cord which has synaptic connections with the dorsal horn interneurons,

are primarily stimulated by PAG. The chemical 5-HT is the main neurotransmitter at these

synapses. Transmission in nociceptive pathways is then inhibited when the pathway from the

NRM to the substantia gelatinosa of the dorsal horn is activated due to the increase in the 5-HT

levels (Duman, et al., 2004).

Antinociception induced by aspartame, involving opioidergic mechanism, could be a

potential therapy for acute pain. It can be an alternative for sucrose in providing sweet-taste

analgesia to obese patients who require a low calorie diet. However, since aspartame contains

phenylalanine, it cannot be used on patients with phenylketonuria wherein elevation of blood

phenylalanine can be dangerous. Further studies are still needed in order to discover the actual

therapeutic place of aspartame in pain therapy for humans.


19

CONCLUSION

Aspartame-induced antinociception via supraspinal and spinal structures involves

opioidergic mechanisms. In addition, the effect of aspartame was consistent in all three trial runs

in the present investigation. As far as the present findings are concerned, aspartame would

exhibit an antinociceptive effect following a relatively long period of administration of

treatment.

Furthermore, for a brief duration wherein treatment was administered for one day,

naloxone in the absence of aspartame administration can induce temporary antinociception in

mice. Since the observed antinociceptive effect of naloxone is very transient, it is not an ideal

alternative for pain medication. Reduction in pain response produced by aspartame could be

used as potential therapy for acute pain, which may be an alternative for sucrose in providing

sweet-taste analgesia to obese patients who require a low calorie diet.


20

RECOMMENDATIONS

The possible areas for further investigation involving aspartame antinociception are the

following: first, the treatment (aspartame and control vehicle) should be administered with the

use of a gavage in order to ensure an even administration of treatment. Second, a positive control

group such as mefenamic acid may be included in the experiment for comparison with

aspartame. Third, the sample size should be increased to guarantee statistical precision of

experimental design. Fourth, the antinociceptive effect of aspartame should also be tested in a

chronic pain model wherein nociception is persistent such as carrageenan-induced inflammation

to assess whether aspartame can be used for pain relief in arthritis and other related health

problems. Lastly, other opioid receptor antagonists such as naltrindole and nor-binaltorphimine

should be used to identify the specific opioid receptor involved in aspartame-induced

antinociception, whereas proglumide and theophylline should be used in order to uncover other

possible mechanisms by which aspartame induce antinociception.


21

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Abdollahi, M., J. Sarrafzadeh, S. Nikfar and F. Roshanzamir. 2003. Mechanism of aspartame-induced antinociception in mice. Indian J. Pharmacol. 35: 37-41.

Atamer-Simsek, S., H. Olmez-Salvarli, O. Guc and L. Eroglu. 2000. Antinociceptive effect of amikacin and its interaction with morphine and naloxone. Pharmacol. Res. 41: 355-360.

Chapman, C.R. and J.D. Loeser. 1989. Issues in pain measurement. Raven Press. New York.

Diaz-Reval, M.I., R. Ventura-Martinez, M. Deciga-Campos, J.A. Terron, F. Cabre and F.J. Lopez-Munoz. 2002. Involvement of serotonin mechanisms in the antinociceptive effect of S(+)-ketoprofen. Drug Dev. Res. 57: 187-192.

Duman, E.N., M. Kesim, M. Kadioglu, E. Yaris , N.I. Kalyoncu and N. Erciyes. 2004. Possible involvement of opioidergic and serotonergic mechanisms in antinociceptive effect of paroxetine in acute pain. J. Pharmacol. Sci. 94: 161-165.

Ferrara, P. and C.H. Li. 1980. B-endorphin: characteristics in binding sites of rabbit spinal cord. Proc Natl Acad Sci. 77: 5746-5748.

Golan, D.E. 2007. Principles of pharmacology: The pathophysiologic basis of drug therapy. Lippincott Williams and Wilkins. Maryland.

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Johnston, C.C., et al. 2002. Routine sucrose analgesia during the first week of life in neonates younter than 31 weeks’ postconceptional age. Pediatrics 110: 523-528.

Kruger, L. 2001. Methods in pain research. CRC Press. Florida.

Law, P., H.H. Lo and C.H. Li. 1979. Properties and localization of β-endorphin receptor in rat brain. Proc. Natl. Acad. Sci. 76: 5455-5459.

Lean, M.E.J. and C.R. Hankey. 2004. Aspartame and its effects on health. Br. Med. J. 329: 755-756.


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Nakashima, K. and Y. Ninomiya. 1998. Increase in inositol 1,4,5-triphosphate levels of the fungiform papilla in response to saccharin and bitter substances in mice. Cell Physiol. Biochem. 8: 224-230.

Nikfar, S., M. Abdollahi, F. Etemad and M. Sharifzadeh. 1997. Effects of sweetening agents on morphine-induced antinociception in mice by formalin test. Gen. Pharmacol. 29: 583-586.

Ong, B. and R. Segstro. 1994. Respiratory depression associated with meperidine spinal anaesthesia. Can. J. Anaesth. 41: 725-727.

Pepino, M.Y. and J.A. Mennella. 2005. Sucrose-induced analgesia is related to sweet preferences in children but not adults. J. Pain. 119: 210-218.

Porth, C. Pathophysiology: Concepts of altered health states. 7th ed. Lippincott Williams and Wilkins. Maryland.

Ramenghi, L.A., D.J. Evans and M.I. Levene. 1999. “Sucrose analgesia”: Absorptive mechanism or taste perception? Arch. Dis. Child Fetal Neonatal Ed. 80: 146-147.

Ren, K., E.M. Blass, Q-q. Zhou and R. Dubner 1997. Suckling and sucrose ingestion suppress persistent hyperalgesia and spinal Fos expression after forepaw inflammation in infant rats. Proc. Natl. Acad. Sci. 94: 1471-1475.

Sauro, M.D. and R.P. Greenberg. 2005. Endogenous opiates and the placebo effect: A meta-analytic review. J. Psychosom. Res. 58: 115-120.

Segato, F.N., C. Castro-Souza, E.N. Segato, S. Morato and N.C. Coimbra. 1997. Sucrose ingestion causes opioid analgesia. Braz. J. Med. Biol. Res. 30: 981-984.

Sharma, S., N.K. Jain and S.K. Kulkarni. 2005. Possible analgesic and anti-inflammatory interactions of aspartame with opioids and NSAIDs. Indian J. Exp. Biol. 43: 498-502.

Shigeru, A., A. Kazuhito, H. Hiroyuki, H. Tadashi and S. Michio. 2003. Enhancement of beta-endorphin levels in rat hypothalamus by exercise. Jpn. J. Phys. Fitness Sports Med. 52: 159-166.

South, S.M. and M.T. Smith. 1998. Apparent insensitivity of the hotplate latency test for detection of antinociception following intraperitoneal, intravenous or intracerebroventricular M6G administration to rats. J. Pharmacol. Exp. Ther. 286: 1326-1332.

Stegink, L.D., L.J. Filer, G.L. Baker and J.E. Mcdonnell. 1980. Effect of an abuse dose of aspartame upon plasma erythrocyte levels of amino acids in phenylketonuric heterozygous and normal adults. Nutrition 110: 2216-2224.


23

Stegink, L.D., L.J. Filer, E.F. Bell, and E.E. Ziegler. 1987. Plasma amino acid concentrations in normal adults administered aspartame in capsules or solution: lack of bioequivalence. Metabolism 36: 507-512.

Tabarelli, Z., D.B. Berlese, P.D. Sauzem, C.F. Mello and M.A. Rubin. 2003. Antinociceptive effects of Cremophor EL orally administered to mice. Braz. J. Med. Biol. Res. 36: 119-123.

Taddio, A., V. Shah, P. Sha and J. Katz. 2003. Β-endorphin concentration after administration of sucrose in preterm infants. Arch. Pediatr. Adolesc. Med. 157: 1071-1074.

Tsuruoka, M., Y. Hiruma, K. Matsutani and Y. Matsui. 1998. Effects of yohimbine on naloxone-induced antinociception in a rat model of inflammatory hyperalgesia. Eur. J. Pharmacol. 348: 161-165

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Yellowlees H. 1983. Aspartame. Br. Med. J. 287: 912-913.


24

TABLES


25

Table 1. Mean weights of (±S.E.M.) mice negative control groups and aspartame-treated groups.

Treatment Groups Initial Weight Weight at Day 15 Weight at Day 28

AS 18.84167 ± 0.90942 24.34167 ± 0.95374 27.85000 ± 1.06867

WS 18.16667 ± 0.75321 22.67500 ± 1.08321 27.39091 ± 1.08321

AN 17.71667 ± 0.81861 22.95000 ± 0.62468 26.69167 ± 0.67974

WN 17.52500 ± 0.96767 23.21667 ± 0.86013 26.98333 ± 0.068753W, water; A, aspartame; S, saline, N, naloxone.

Table 2. Mean amount of aspartame solution consumed by mice treatment groups.

Treatment Groups Amount of Aspartame solution consumed (ml)

AS 14.847045 ± 2.18144

AN 11.92449 ± 1.06454A, aspartame; S, saline, N, naloxone.

Table 3. Mean index of analgesia (±S.E.M.) of mice negative control groups and aspartame-treated groups.

Duration of Administration of Treatment

Water AspartameSaline Naloxone Saline Naloxone

1 day -0.46845 ± 0.34076

0.16445 ± 0.16055

-0.08608 ± 0.06012

-0.12643 ± 0.24182

14 days -0.64620 ± 0.38005

-0.19701 ± 0.22795

-0.02777 ± 0.11212

-0.40277 ± 0.27306


26

Table 4. Mean differences of mice negative control groups and aspartame-treated groups.

95% Confidence IntervalTreatment Groups Mean Difference Lower Bound Upper boundAS (1-d) versus WS (1-d) 0.38237 -0.00321 0.76795AS (14-d) versus WS (14-d) 0.61843§ 0.17688 1.05998AS (1-d) versus AN (1-d) 0.04035 -0.23732 0.31801AS (14-d) versus AN (14-d) 0.37499§ 0.04606 0.70393WS (1-d) versus WN (1-d) 0.63290§ 0.21315 1.05265WS (14-d) versus WN (14-d) 0.44919 -0.04465 0.94303W, water; A, aspartame; S, saline, N, naloxone; 1-d, one day; 14-d, 14 days.§Mean difference is significant at α = 0.05


27

FIGURES


28

Figure 1. Mean index of analgesia of mice negative control groups and aspartame-treated groups. W, water; A, aspartame; S, saline, N, naloxone. Range bars represent S.E.M. for 12 mice in each group. *T = differences in treatment effect are significant; *A = differences in antagonist effect are significant.

Figure 2. Mean index of analgesia of mice negative control groups and aspartame-treated groups comparing the effect of aspartame after one and 14 days of administration. W, water;


29

A, aspartame; S, saline, N, naloxone. Range bars represent S.E.M. for 12 mice in each group. *T = differences in treatment effect are significant.

Figure 3. Mean index of analgesia of mice aspartame-treated groups comparing the effect of naloxone after one and 14 days of administration of treatment. A, aspartame; S, saline, N, naloxone. Range bars represent S.E.M. for 12 mice in each group. *A = differences in antagonist effect are significant.

Figure 4. Mean index of analgesia of mice negative control groups comparing the effect of naloxone after one and 14 days of administration of treatment. W, water; S, saline, N,


30

naloxone. Range bars represent S.E.M. for 12 mice in each group. *A = differences in antagonist effect are significant.

APPENDICES


31

Appendix A1. Structure of Aspartame (N-L-aspartyl-L-phenylalanine-1-methyl ester)

Appendix A2. Structure of Naloxone molecule


32

Appendix B. Table of analysis of variance for within subject effects.

Source

Type III Sum of Squares df

Mean Square F P

Duration 1.072 1 1.072 18.879 .000

Duration * Treatment .104 1 .104 1.824 .192

Duration * Test .974 1 .974 17.153 .001

Duration * Naloxone .039 1 .039 .696 .414

Duration * Run .032 2 .016 .278 .760

Duration * Treatment * Test .004 1 .004 .066 .800

Duration * Treatment * Naloxone .250 1 .250 4.400 .049

Duration * Test * Naloxone .062 1 .062 1.098 .307

Duration * Treatment * Test * Naloxone .233 1 .233 4.103 .056

Duration * Treatment * Run .365 2 .182 3.212 .062

Duration * Test * Run .115 2 .058 1.016 .380

Duration * Treatment * Test * Run .271 2 .136 2.387 .118

Duration * Naloxone * Run .264 2 .132 2.325 .124

Duration * Treatment * Naloxone * Run .075 2 .037 .660 .528

Duration * Test * Naloxone * Run .544 2 .272 4.787 .020

Duration * Treatment * Test * Naloxone * Run

.140 1 .140 2.464 .132

Error(Duration) 1.135 20 .057


33

Appendix C. Mean values of index of analgesia (± S.E.M.) of mice treatment groups(for all trial runs)

RunType of

Behavioral Test

1-Day 14-DayWater Aspartame Water Aspartame

Naloxone Saline Saline Naloxone Naloxone Saline Saline Naloxone

First

Hot plate 1.00±0.00

-1.62±0.10

0.07±0.20

0.58±0.42

0.16±0.01

-2.68±.

0.40±0.15

-0.46±0.17

Tail flick 0.04±0.10

-0.01±0.00

0.06±0.08

0.25±0.24

-0.01±0.08

-0.22±0.14

0.06±0.05

0.69±0.31

Second

Hot plate . -3.15±.

-0.32±.

-1.66±0.74

. -3.51±.

-1.04±.

-1.95±1.00


-0.03±0.05

0.06±0.01

0.06±0.02

0.02±0.05

-0.13±0.03

0.00±0.05

-0.13±0.05

Third

Hot plate -0.22±0.68

0.70±0.28

-0.23±0.13

0.11±0.00

-1.12±1.31

0.20±0.08

-0.02±0.05

-0.39±0.25


-0.03±0.03

-0.15±0.24

-0.10±0.04

-0.04±0.01

0.07±0.04

-0.18±0.14

-0.07±0.08

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