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Neurotropic Effects of Aspartame, Stevia and Sucralose on Memory Retention and on the Histology of the Hippocampus of the ICR Mice (Mus Musculus)

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Neurotropic Effects of Aspartame, Stevia and Sucralose on Memory Retention and on the Histology of the Hippocampus of the ICR Mice (Mus Musculus) 114 Asian Pac J Trop Biomed 2016; 6(2): 114–118 HOSTED BY Contents lists available at ScienceDirect Asian Pacific Journal of Tropical Biomedicine journal homepage: www.elsevier.com/locate/apjtb Original article http://dx.doi.org/10.1016/j.apjtb.2015.11.001 Neurotropic effects of aspartame, stevia and sucralose on memory retention and on the histology of the hippocampus of the ICR mice (Mus musculus) Lejan Miguel Alabastro Villareal, Rachelle Anne Montes Cruz, Michael Bagui Ples, Rodel Jonathan Santos Vitor II* Biology Department, College of Science, De La Salle University, 2401 Taft Avenue, 0922 Manila, Philippines ARTICLE INFO ABSTRACT Article history: Objective: To identify the effects of the consumption of non-nutritive sweeteners on Received 13 Apr 2015 memory retention and on the histology of the hippocampus. Received in revised form 26 Oct 2015 Methods: In this study, 20 mice were used to determine if there is an effect of Accepted 10 Nov 2015 consuming the maximum allowable dose of the non-nutritive sweeteners on the memory Available online 10 Dec 2015 retention and on the histology of the hippocampus. The mice were distributed into four groups and the treatments were given via oral gavage: Group 1 (water), Group 2 (aspartame: 1000 mg/kg), Group 3 (stevia: 1000 mg/kg) and Group 4 (sucralose: Keywords: 16000 mg/kg). Treatments were administered to the different experimental groups for 32 Aspartame days, after which memory retention was tested using the two-day water maze protocol. Hippocampus After the tests, the mice were sacrificed and the brain was analyzed histologically for Learning neurotrophic effects. Memory Results: Based on the results of the two-day water maze protocol, there were no dif- Mus musculus ferences between the non-nutritive sweeteners and the control group. However, stevia Stevia showed high cellular apoptosis followed by aspartame, sucralose and control group. Sucralose Conclusions: There was no significant effect on the memory of the mice. It showed histologically however, that stevia had a significant neurotropic effect compared to the other sweeteners. 1. Introduction aspartame consumption resulted to the longer time for the mice to locate the reward within the T-maze, which showed There are two kinds of sweeteners, namely, nutritive and impaired long-term memory retention [9–11]. non-nutritive sweeteners [1]. Nutritive sweeteners are naturally Stevia as sweeteners has been used for a long time in South occurring like sucrose and fructose [2]. On the other hand, America without any reported adverse effects. Nonetheless, its non-nutritive sweeteners are synthetically made like aspartame, safety has been the subject of controversy for many years [12]. stevia and sucralose [3]. Non-nutritive sweeteners, also referred The metabolically activated steviol has been found to be to as high intensity sweeteners, are typically used in small mutagenic in mice but its activation in humans has not yet amounts to reduce the caloric intake while sustaining the desired been established with further work needed to establish its taste in many food products [4]. toxicity in humans [13]. According to the study done by Romano et al., some of the Sucralose is one of the newest non-nutritive sweeteners and its adverse effects on the central nervous system caused by the safety has been established by different experiments [14–17]. intake of aspartame are headaches, mood changes, insomnia and However, according to Schiffman and Rother, experiments in rat seizures [5]. In addition, other effects include confusion, models showed that consumption of foods and fluids containing personality disorders, dizziness and visual difficulty [6]. high-potency sweeteners was interfered with the ability to detect Recently, it has also been attributed to obesity [7,8]. Chronic sweet tastants, thereby affecting energy regulation [17]. *Corresponding author: Rodel Jonathan Santos Vitor II, DVM, Assistant Professorial Lecturer, Biology Department, De La Salle University, 2401 Taft Avenue, 0922 Manila, Philippines. Tel: +63 2 524 4611 ext. 460, +63 917 803 2687 Fax: +63 2 536 0228 E-mail: [email protected] All experimental procedures involving animals were conducted in accordance to PALAS Code of Practice for the Care and Use of Laboratory Animals in the Philippines and approved by the Institutional Animal Care and Use Committee of De La Salle University. Peer review under responsibility of Hainan Medical University. The journal implements double-blind peer review practiced by specially invited international editorial board members. 2221-1691/Copyright © 2016 Hainan Medical University. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/). Lejan Miguel Alabastro Villareal et al./Asian Pac J Trop Biomed 2016; 6(2): 114–118 115 Despite the adverse effects caused by some artificial sweet- rest days for all the mice. The platform was randomly placed for eners, they are still widely used and accepted all over the world the Test 1 and then changed for Test 2 [22]. [18]. Results of this study can be used to reevaluate the maximum daily dose of artificial sweeteners and for further studies on the 2.3.1. Training phase (Day 1) toxicity of aspartame, stevia and sucralose. The platform was randomly placed within the pool with the pre-release points at north, south, east, or west of the pool. These 2. Materials and methods pre-release points had signs that north, south, east or west were attached to the wall of the pool. In addition, the distance from the 2.1. Animal use and maintenance pre-release points to the randomly placed platform was measured and recorded. The animals were released facing the wall and a A total of 20, 6-week old, male ICR mice (Mus musculus) maximum of 60 s was allowed for the mice to locate the platform. were procured from the Research Institute for Tropical Medi- If the mice failed to locate the platform within the allotted time, cine, Alabang, Muntinlupa City. They were randomly distrib- they were guided to the hidden platform and placed there for 15 s, uted to the four groups (n = 5), namely, control group (Group 1), for them to learn that the safe zone was the platform, before they aspartame (Group 2), stevia (Group 3) and sucralose (Group 4). were returned to the cages. If the animals jumped off the platform, The mice were kept in individual cages in the animal house of the animals were guided back towards the platform. The time was De La Salle University. All cages were cleaned and sanitized recorded when the mouse found the hidden platform and if they weekly and bedded with husk which was autoclaved. The plates failed, the recorded time was 1 min. This was done two times to and water bottles were cleaned and dried twice a week. The mice each mouse (two trials) with intertrial time of 30 s. were allowed to acclimatize for one week to adjust to a 12 h light: 12 h dark cycle at 24–26 C. Food and water were given 2.3.2. Test phase (Day 2) ad libitum. Proper handling and maintenance of the mice were The platform randomly placed in Day 1 was retained. The guided by the safety standards set by the Philippine Association animals were still released facing the wall and a maximum of of Laboratory Animal Science as approved by the De La Salle 60 s was allowed for the mice to locate the platform submerged fi University (Institutional Animal Care and Use Committee). underwater. However, if the animals failed to nd the hidden platform within 60 s, they were guided to the platform and 2.2. Treatment preparation and administration placed there for 15 s, before returning them back to the cages. If the animals jumped off the platform, the animals were guided Artificial sweeteners (aspartame, stevia and sucralose) were back towards the platform. purchased from local supermarkets and were dissolved in 0.2 mL of distilled water based on the amount to be given into 2.4. Histological analysis per mouse (Table 1). About 0.2 mL of the different treatments was administered to the mice via gastric gavage for 32 days [19]. After 32 days of experiment, the mice were euthanized by cervical dislocation and the brain was extracted and placed in a vial containing 10% buffered formalin. Samples were then Table 1 brought to the Pathology Department, College of Medicine, Treatment given to the different groups for duration of 28 days. University of the Philippines Manila for brain processing and Groups Dose Reference routine hematoxylin and eosin slide preparation. Control 0.2 mL distilled water The processed hippocampal sections of the brain were Aspartame 1000 mg/kg body weight in 0.2 mL [20] analyzed by counting the normal cells and the cells that under- distilled water gone apoptosis per high power objective. The person that Stevia 1000 mg/kg body weight in 0.2 mL [13] distilled water counted the neurons was blinded to the experimental group of Sucralose 16000 mg/kg body weight in 0.2 mL [16] the particular sample being examined and did all the neuronal distilled water counts to avoid Inter-counter variation. 2.5. Statistical analysis 2.3. Two-day water maze protocol The differences in rate and number of neuronal cells were According to Hodges, the water maze, which was used for analyzed using One-way ANOVA and the means were compared determining memory retention in mice, was tapped into the using Tukeys test and SPSS version 22 was used to determine cognitive processes that included associative learning, short- significant differences among the treatment groups at P < 0.05. term and long-term non-spatial memory, temporal order and conditional discrimination or anxiety [21].
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