Aspartame (ASP) is an artificial sweetener used in food products as alternative to sugar. Because aspartame is approximately 200 times sweeter than sugar, it is used in low-calorie soft drinks and foods . Aspartame consists of two amino acids (aspartic acid and phenylalanine) and methanol. It was approved by Food Drug Administration (FDA) for use in dry applications in 1981, followed by approval for use in carbonated soft drinks in 1983 and as a general sweetener in 1996 . It is now known that aspartame represents 62% of the value of the intense sweetener market in terms of its world consumption , for example, in the US, more than 70% of aspartame sales are attributed to soft drinks . Upon ingestion, aspartame is metabolized by digestive esterases and peptidases in the intestinal lumen to methanol and its constituent amino acids . Subsequent to aspartame consumption, the concentrations of its metabolites are increased in the blood . Ever since its approval by the FDA for use as an artificial sweetener, aspartame has been the subject of much debate with respect to its health effects such as increasing brain cancer rates . It has also been reported that high doses of aspartame can also generate major neurochemical changes in rats [8, 9]. The toxicity of aspartame has been linked to its pro-oxidative effects in animal studies . Generation of excessive reactive oxygen species (ROS) by aspartame has led to a marked increase in pro-apoptotic marker (Bax), whereas decrease in anti-apoptotic marker (Bcl-2) in rats’ brain, indicating that aspartame is harmful at cellular level . ROS are generated continuously during oxidative metabolism in cells. Because of their metabolic rate, vital biochemical functions and high content of oxidizable substrates, some vital organs in the body are very vulnerable to ROS. To prevent the effects of ROS, organisms have evolved multiple systems of antioxidant defence that are essential for cellular metabolism and functions . On the basis of the aforementioned, this study was undertaken to investigate the effects of long-term administration of aspartame on redox status, lipid profile and biochemical indices in kidney, liver and brain of male Wistar rats.
Materials and methods
Aspartame was purchased from a pharmacy store in Berlin, Germany. Thiobarbituric acid (TBA) was procured from Aldrich Chemical Co. (Milwaukee, WI, USA). Glutathione, hydrogen peroxide, 5,5′-dithios-bis-2-nitrobenzoic acid (DNTB) and epinephrine were purchased from Sigma Chemical Co. (Saint Louis, MO USA). Other reagents were of analytical grade and the purest quality available.
Inbred male Wistar rats weighing between 185 and 193 g were purchased from the animal house of the Department of Veterinary Physiology, Biochemistry and Pharmacology, University of Ibadan, Nigeria. These animals were kept in ventilated cages at room temperature (28 °C–30 °C) and maintained on normal laboratory chow (Ladokun Feeds, Ibadan, Nigeria) and water ad libitum. Rats handling and treatments conform to guidelines of the National Institute of Health (NIH publication 85-23, 1985) for laboratory animal care and use. The study was approved by the Animal Ethics Committee, Faculty of Basic Medical Sciences, University of Ibadan, Nigeria.
Twenty adult male Wistar rats were randomly divided into four groups of five rats each. The 1st group (control) received drug vehicle (distilled water), whereas the 2nd, 3rd and 4th groups tagged ASP 1, ASP 2 and ASP 3 received ASP at doses of 15, 35 and 70 mg/kg body weight, respectively. ASP was given once daily by oral gavage for consecutive 9 weeks.
Preparation of tissues
The rats were fasted overnight and sacrificed 24 h after the last dose of ASP. Kidney, liver and brain were quickly removed and washed in ice-cold 1.15% KCl solution to remove blood stains, dried and weighed. The tissues were homogenized in four volumes of 50 mM phosphate buffer, pH 7.4 and centrifuged at 10,000 g for 15 min to obtain post-mitochondrial supernatant fraction (PMF). Procedures were carried out at temperature of 4 °C.
Preparation of serum
Blood was collected from the heart of the animals into plain centrifuge tubes and was allowed to stand for 1 h. Serum was prepared by centrifugation at 3000 g for 15 min in a Beckman bench centrifuge (Beckman and Hirsch, Burlington, IO, USA). The clear supernatant was used for the estimation of serum lipids and enzymes.
Protein contents of the samples were assayed by the method of Lowry et al. , using bovine serum albumin as standard. The activities of alanine and aspartate aminotransferases (ALT and AST) were assayed by the combined methods of Mohun and Cook , and Reitman and Frankel . Serum gamma-glutamyl transferase (GGT) and lactate dehydrogenase (LDH) activities were determined by the methods of Fossati et al. , and Zimmerman and Weinstein , respectively. Serum total cholesterol level was assayed by the method of Richmond et al. . The method involved enzymatic hydrolysis and oxidation of cholesterol with the formation of quinone imine (an indicator) from hydrogen peroxide and 4-aminoantipyrine in the presence of phenol and peroxide. The serum level of triglyceride was determined by Jacobs and Van Demark , and Koditschek and Umbreit ; this was based on the hydrolysis of triglycerides with the formation of glycerol which is substrate for other enzymes with the subsequent formation of hydrogen peroxide. This then reacts with 4-aminophenazone and 4-chlorophenol in the presence of peroxidase to give quinoneimine which is measured spectrophotometrically at 500 nm. The lipoproteins (measured using the enzymatic colorimetric method), very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL) were precipitated by the addition of phosphotungstic acid and magnesium chloride. After centrifugation at 3000 g for 10 min at 25 °C, the clear supernatant contained HDL fraction, which was assayed for cholesterol with the Randox diagnostic kit. The LDL was calculated using the formula of Friedewald et al. . Lipid peroxidation level was assayed by the reaction between 2-thiobarbituric acid (TBA) and malondialdehyde (MDA), an end product of lipid peroxides as described by Buege and Aust . Superoxide dismutase (SOD) activity was measured by the nitro blue tetrazolium (NBT) reduction method of McCord and Fridovich . Catalase (CAT) activity was assayed spectrophotometrically by measuring the rate of decomposition of hydrogen peroxide at 240 nm as described by Aebi . Reduced glutathione level was measured by the method of Beutler et al. ; this method is based on the development of a relatively stable (yellow) colour when 5′,5′-dithiobis-(2-nitrobenzoic acid) (Ellman’s reagent) is added to sulfhydryl compounds. The chromophoric product resulting from the reaction of Ellman’s reagent with the reduced glutathione (2-nitro-5-thiobenzoic acid) possesses a molar absorption at 412 nm which is proportion to the level of reduced glutathione in the test sample. The glutathione peroxidase (GPx) activity was assessed by the method of Rotruck et al. , whereas glutathione-S-transferase (GST) activity was determined according to Habig et al. ; the principle is based on the fact that all known GST demonstrate a relatively high activity with 1-chloro-2,4-dinitrobenzene as the 2nd substrate. When this substance is conjugated with reduced glutathione, its absorption maximum shifts to a longer wavelength 340 nm and the absorption increase at this wavelength provides a direct measurement of the enzymatic reaction.
All values were expressed as the mean±SD of five animals per group. Data were analysed using one-way ANOVA followed by the post-hoc Duncan multiple range test for analysis of biochemical data using SPSS (10.0; SPSS Inc., Chicago, IL, USA). Values were considered statistically significant at p<0.05.
Effects of aspartame on weight and relative weight of organs, and biochemical markers in rats
There were significant increases in the weight of liver (p=0.032; 0.037) and brain (p=0.029; 0.031) in rats treated with ASP 2 and ASP 3 relative to controls. Also, the relative weight of liver significantly increased (p=0.023; 0.027) in ASP 2- and ASP 3-treated rats when compared to controls (Table 1). Administration of ASP 2 and ASP 3 increased the activities of serum alanine and aspartate aminotransferases (ALT and AST) by 1.7-, 1.2- and 2.2-, 1.5-fold, respectively. Furthermore, activities of serum gamma glutamyl transferase (GGT) increased by 70% and 85% in ASP 2- and ASP 3-treated rats, respectively (Table 2). In Table 3, administration of ASP 2 and ASP 3 markedly elevated serum urea and conjugated bilirubin by 72%, 58% and 63%, 64%, respectively, whereas ASP 3 increased serum creatinine level by 57%. However, ASP 1 did not significantly affect the weight of organs (p=0.075–0.093), activities of ALT, AST, GGT and levels of urea, creatinine and conjugated bilirubin (p=0.061–0.11).
Effects of aspartame on redox status or oxidant/antioxidant balance in rats
Administration of ASP significantly increased (p=0.018–0.025) kidney, hepatic and brain lipid peroxidation (LPO) products measured as TBARS in the rats (Figure 1) with concomitant decrease in the levels of reduced glutathione (Figure 2). Treatment of rats with ASP 2 and ASP 3 caused significant (p=0.024–0.041) reduction in the activities of kidney, hepatic and brain superoxide dismutase and glutathione-s-transferase (Figures 3 and 4), whereas only hepatic and brain catalase and glutathione peroxidase activities were adversely affected by ASP 2 and ASP 3 (p=0.031–0.045) (Figures 5 and 6).
Effects of aspartame on lipid profile of rats
Administration of aspartame at doses of 35 and 70 mg/kg/day (ASP 2 and ASP 3) for consecutive 9 weeks significantly increased the levels of serum total cholesterol, triglycerides and LDL-cholesterol by 1.3-, 1.5-, 2.4-fold and 1.8-, 1.4-, 2.5-fold, respectively (p=0.031–0.042). In addition, rats fed on aspartame at a dose of 70 mg/kg/day had their serum HDL-C levels decreased by 33% relative to control. In contrast, aspartame at a dose of 15 mg/kg/day (ASP 1) did not elicit adverse affects on the lipid profile of the rats (Table 4).
Effects of aspartame on the cyto-architecture of brain, liver and kidney of rats
Figure 7 indicates that the brain tissues from control group have normal architecture with no visible lesions. However, brain tissues from ASP 2 and ASP 3 groups contained sub-dural and focal hemorrhagic lesions, whereas mild lesions were seen in the brain of rats given ASP 1. In Figure 8, no visible lesions were seen in liver tissues from control and ASP 1 groups. Administration of ASP 2 and ASP 3 caused severe necrosis, periportal infiltration, congestion and dilatation of sinusoids of the hepatocytes. Figure 9 shows adequate, well-preserved glomeruli and normal architecture of kidney tissues in the control and ASP 1 groups. However, ASP 2 and ASP 3 caused severe congestion of vessel in the cortex and medulla as well as hyperplasia of the cuboidal cells of the renal tissues.
The present results give further data to support the idea that aspartame may induce redox imbalance, altered biochemical indices and lipid profile in rats after long-term administration. The findings were supported by both biochemical and histological data. Aspartame is widely used as an artificial sweetener in low-calorie foods and soft drinks. Several tests on the safety and toxicity of aspartame have been performed and the results are conflicting. The toxicity of any agent is based on the fact that toxicity increases in a dose-dependent manner and that explains the reason for using three dose of aspartame in this study (15, 35 and 70 mg/kg). The aspartame doses used in this study are very relevant to human. The acceptable daily intake levels of aspartame established by European Food Safety Authority and US Food and Drug Administration are 40 and 50 mg/kg/day, respectively . The main question is whether aspartame could be harmful at common or abuse usage levels. The first carcinogenetic effect of aspartame in an animal model was reported by Soffritti et al.  using bioassays at a dose of 40 mg/kg body weight/day, which falls within the doses chosen for this study. Abdel-Salam et al.  reported that the administration of aspartame alone or in the presence of a mild systemic inflammatory response increases oxidative stress and inflammation in the brain. This observation was consistent with the findings of the present study, in which aspartame at all doses increased lipid peroxidation (LPO) products in the kidney, liver and brain of rats. The finding derives its importance from the fact that increased oxidative stress has been linked to several ailments such as diabetes, cancer, neurodegenerative diseases or other age-related cognitive deficits [31, 32]. The prime targets for free radical reactions during aspartame metabolism are the unsaturated bonds in membrane lipids. Consequent peroxidation results in a loss in membrane fluidity and receptors alignment, suggesting oxidative damage to macromolecules such as lipids . Oxidative stress can be the result of increased free radicals production or alternatively decreased endogenous antioxidants. In this study, we found that the levels of reduced glutathione and activities of antioxidant enzymes such as superoxide dismutase, catalase, glutathione-s-transferase and glutathione peroxidase were significantly reduced in the liver and brain of animals treated with ASP 2 and ASP 3. This observation is consistent with the findings of Abhilash et al. . GSH is an important non-enzymatic antioxidant that plays a critical role in the maintenance of thiol redox potential in cells, keeping sulfhydryl groups of cytosolic protein in the reduced form, and plays a crucial role in the detoxification of toxic chemicals of endogenous and exogenous origin. GSH directly reacts with radicals in non-enzymatic reactions and is the electron donor of peroxides catalyzed by glutathione peroxidase. In addition to free radical produced during aspartame metabolism causing oxidative stress and depletion of antioxidant enzymes, Kruse  reported that among the aspartame metabolites, methanol is the toxicant that causes systemic toxicity. The primary metabolic fate of methanol is the direct oxidation to formaldehyde and then into formate. Skrzydlewska  revealed that cellular glutathione content and activity of glutathione-dependent enzymes were decreased in methanol intoxication which is the hall mark of aspartame toxicity. Therefore, decreased levels of GSH or activities of GSH-dependent enzymes observed in this study may be as a result of the damaging effect of free radicals produced after methanol exposure or could be a direct effect of formaldehyde formed from the oxidation of methanol on these enzymes. In this study, treatment of rats with aspartame at doses of 35 and 70 mg/kg caused significant increase in the levels of liver and kidney function markers such as creatinine, blood urea nitrogen and conjugated bilirubin relative to controls, thus indicating hepato-renal dysfunction. These injuries could be due to production of free radicals or involvement of oxidative stress during aspartame metabolism. Aspartame may influence the various metabolic pathways of liver thereby enhancing the levels of total cholesterol and triglycerides as noticed in the present study. Also, oxidative damage to kidney by aspartame may contribute to the observed high concentrations of blood urea nitrogen and creatinine in serum. Similar results have been reported in earlier studies where it was suggested that kidney dysfunction due to ASP administration occurred via oxidative stress . The adverse effects of ASP on kidney as observed in this study may be detrimental to health of diabetic subjects that consume aspartame regularly in soft drinks. It is known that prolonged and untreated diabetes are characterised by high serum urea and creatinine . Based on the present study, consumption of ASP by diabetic patients will further aggravate the health conditions of these individuals. Aspartame at doses of 35 mg/kg and above caused hepatotoxicity as clearly indicated by the significant increase in levels of ALT, AST and GGT. Serum levels of transaminases is generally considered as sensitive markers of liver function and their concentrations are increased in the serum because of their cytoplasmic nature and are thus released into blood due to changes in the permeability of hepatocyte membranes. Increased level of LDH in serum in the present investigation apparently indicated the toxic effects of aspartame at doses of 35 and 70 mg/kg in the rats. Histology results showed the presence of lesions in liver, kidney and brain of rats treated with aspartame at doses of 35 and 70 mg/kg, which correlate with the levels of liver and kidney function biochemical markers in the rats.
In conclusion, we believe that the metabolism of aspartame may induce oxidant/antioxidant imbalance due to free radicals generated by aspartame. Also, depletion of both enzymic and non-enzymic antioxidant parameters observed in the tissues of the rats after long-term intake of aspartame is a clear sign of oxidative stress. It has been observed that in addition to the population with diabetes mellitus, children and pregnant women are among the major consumers of aspartame; these findings may likely have important health implications because of the widespread use of aspartame in foods and beverage preparations. However, this is a single study with few animals, therefore, more studies are necessary to confirm the present effects, as well as, the mechanisms responsible for the aspartame toxicity.
The authors are grateful to the technical assistance of Messers E. Sabo and O.K. Ajiboye of the Department of Biochemistry, University of Ibadan, Nigeria.
Author contributions: The authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.
Research funding: None declared.
Employment or leadership: None declared.
Honorarium: None declared.
Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis and interpretation of data; in the writing of the report or in the decision to submit the report for publication.
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About the article
Published Online: 2015-08-06
Published in Print: 2016-01-01