Effect of long term intake of aspartame on antioxidant defense status in liver

Effect of long term intake of aspartame on antioxidant defense status in liver

Food and Chemical Toxicology 49 (2011) 1203–1207 Contents lists available at ScienceDirect Food and Chemical Toxicology journal homepage: www.elsevi...

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Food and Chemical Toxicology 49 (2011) 1203–1207

Contents lists available at ScienceDirect

Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox

Effect of long term intake of aspartame on antioxidant defense status in liver M. Abhilash, M.V. Sauganth Paul, Mathews V. Varghese, R. Harikumaran Nair ⇑ School of Biosciences, Mahatma Gandhi University, Kottayam, Kerala 686560, India

a r t i c l e

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Article history: Received 19 October 2010 Accepted 25 February 2011 Available online 3 March 2011 Keywords: Aspartame Liver Hepatocellular injury Histopathology Glutathione

a b s t r a c t The present study evaluates the effect of long term intake of aspartame, the artificial sweetener, on liver antioxidant system and hepatocellular injury in animal model. Eighteen adult male Wistar rats, weighing 150–175 g, were randomly divided into three groups as follows: first group was given aspartame dissolved in water in a dose of 500 mg/kg b.wt.; the second group was given a dose of 1000 mg/kg b.wt.; and controls were given water freely. Rats that had received aspartame (1000 mg/kg b.wt.) in the drinking water for 180 days showed a significant increase in activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and c-glutamyl transferase (GGT). The concentration of reduced glutathione (GSH) and the activity of glutathione peroxidase (GPx), and glutathione reductase (GR) were significantly reduced in the liver of rats that had received aspartame (1000 mg/kg b.wt.). Glutathione was significantly decreased in both the experimental groups. Histopathological examination revealed leukocyte infiltration in aspartame-treated rats (1000 mg/kg b.wt.). It can be concluded from these observations that long term consumption of aspartame leads to hepatocellular injury and alterations in liver antioxidant status mainly through glutathione dependent system. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Sweeteners are paid special attention among food additives as their use enables a sharp reduction in sugar consumption and a significant decrease in caloric intake while maintaining the desirable palatability of foods and soft drinks (Vences-Mejia et al., 2006). Sweeteners are also of primary importance as part of nutritional guidance for diabetes, a disease with increasing incidence in developing as well as developed countries (Gougeon et al., 2004). Aspartame (L-aspartyl L-phenylalanine methylester) is a dipeptide artificial sweetener that is widely used as a non-nutritive sweetener in foods and drinks. Aspartame is used as a sweetener in food products including dry beverage mixes, chewable multi-vitamins, breakfast cereals, chewing gum, puddings and fillings, carbonated beverages, refrigerated and non-refrigerated ready to drink beverages, yogurt type products and pharmaceuticals (Rencuzogullari et al., 2004). Aspartame represents 62% of the value of the intense sweetener market in terms of its world consumption (Fry, 1999). Upon ingestion, aspartame is immediately absorbed from the intestinal lumen and metabolized to phenylalanine, aspartic acid and methanol (Ranney et al., 1976). Following aspartame consumption, the concentrations of its metabolites are increased in the blood (Stegink, 1987).

⇑ Corresponding author. Tel.: +91 9447260362. E-mail address: [email protected] (R.H. Nair). 0278-6915/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2011.02.019

The metabolism of xenobiotics to a large extent takes place in the liver. The byproducts of such metabolism sometimes are more toxic than the initial substance. This could lead to hepatic damage and the emergence of hepatic disorders (Ishak et al., 1991). These by-products include oxygen containing molecules that damage vital cell components through oxidation (Fernandez-Checa et al., 1997). They can produce deleterious effects by reacting with complex cellular molecules such as lipids, proteins and DNA. In order to prevent the potential effects of reactive oxygen species (ROS), organisms have evolved multiple systems of antioxidant defense including both enzymatic and non-enzymatic strategy, and are essential for the cellular metabolism and function (Mates, 2000). Although some information is available on the aspartame induced toxicity at various levels (Christian et al., 2004; Simintzi et al., 2007), the studies on the effect of long term oral exposure of aspartame on liver antioxidants are lacking. Moreover, most of the recent studies on aspartame, have been carried out to understand the mechanisms of neurotoxicity (Christian et al., 2004; Tsakiris et al., 2006; Simintzi et al., 2007; Bergstrom et al., 2007) and cancer (Soffritti et al., 2006; Gallus et al., 2007). Despite numerous toxicological studies of aspartame, its effects on hepatic tissue have received little attention. So, there is a need to substantiate whether long term oral consumption of aspartame induces oxidative stress and structural changes in hepatic tissue. We, therefore, investigated the effect of long term oral administration of aspartame on some markers of oxidative stress and hepatocellular injury in male Wistar rats under laboratory

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conditions. In the present study, we aimed to investigate the effects of long term consumption of aspartame on the activities of (1) hepatic marker enzymes – alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and c-glutamyl transferase (GGT) (2) antioxidant enzymes – superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), and glutathione reductase (GR) (3) non-enzymatic antioxidant molecule reduced glutathione (GSH) (4) malondialdehyde (MDA), a marker of lipid peroxidation and (5) liver histopathology. 2. Materials and methods 2.1. Chemicals Aspartame was purchased from Himedia Chemicals, India. Thiobarbituric acid and triton X-100 were purchased from Sigma Aldrich (St. Louis, USA). All other chemicals were purchased from Sisco Research Laboratories (SRL), India. 2.2. Animals Eighteen male adult Wistar rats weighing 150–175 g were purchased from the Small Animal Breeding Station (SABS) of Govt. Veterinary College, Mannuthy, Thrissur, Kerala, India and acclimatized for six days. All the animals were maintained under standard laboratory conditions of temperature (25 °C) and 12 h light and dark cycles throughout the experimental period. The rats were provided with laboratory chow (Hindustan Lever Ltd., India) and tap water adlibitum. Experiments were conducted as per the guidelines of Institutional Animal Ethical Committee, School of Biosciences, Mahatma Gandhi University. 2.3. Experimental protocol Three groups were formed (n = 6) for oral intubations: first group was given aspartame dissolved in water in a dose of 500 mg/kg b.wt.; the second group was given a dose of 1000 mg/kg b.wt.; and controls were given water. After 180 days of treatment, all the animals were euthanized and decapitated, blood was collected and centrifuged at 3000 rpm for 20 min; the clear serum obtained was used for the determination of serum enzymes. Liver was removed immediately, washed in ice cooled 0.15 M NaCl and blotted on a filter paper. Then the tissue was weighed and homogenized by using Teflon glass homogenizer (1/10th weight/volume) in ice cooled tris HCl buffer (0.2 M, pH 7.4). The homogenate was centrifuged at 10,000g for 20 min at 4 °C and the supernatant was used for biochemical assays. 2.4. Biochemical determinations ALT and AST activities in the serum were determined according to the protocol by Reitman and Frankel (1957). ALP in the serum was assayed according to the method of Kind and King (1954). GGT was assayed by the method of Szasz (1969). Malondialdehyde produced during lipid peroxidation was assayed according to the method of Beuge and Aust (1978). SOD activity was measured by the method of Kakkar et al. (1984) with slight modification. One unit was taken as the amount of enzyme that gave 50% inhibition of NBT reduction/mg protein. Catalase activity in the sample was measured according to the method of Aebi (1974) by measuring the decrease in absorbance of H2O2 at 240 nm. GPx activity was measured according the method of Rotruck et al. (1973). GR activity was measured according to the method described by Goldberg and Spooner (1983) in which the rate of conversion of GSSG to GSH was estimated by monitoring the oxidation of NADPH in the assay system at 340 nm. GR level was assayed using the method of Ellman (1959) with slight modification. Protein estimation was carried out according to the method of Lowry et al. (1951). 2.5. Histopathology Liver tissue was taken from the eviscerated animals and fixed in 10% formalin. The tissue was embedded in paraffin and sections of 4 lm thickness were cut using microtome and stained with haematoxylin and eosin (H&E) for light microscopic examination. Leukocyte infiltration into the liver was graded visually on a semi quantitative scale: 0, no influx; 1, rare leukocytes (1–3 cells); 2, few leukocytes (1–5 cells); 3, moderate leukocyte infiltration (5–15 cells); 4, marked leukocyte infiltration (greater than 15 cells); and 5, severe inflammatory leukocyte infiltration (Horn et al., 2000). 2.6. Statistical analysis The results were analyzed using statistical programme SPSS/PC+, version 10 (SPSS, Inc., Chicago, IL, USA). One way ANOVA was employed for comparison among the three groups. LSD post hoc multiple comparison test was used to determine significant difference among groups. p < 0.05 was considered significant.

3. Results Aspartame administration (1000 mg/kg b.wt.) significantly increased AST, ALT, ALP and GGT in serum. Significant increase in AST and GGT was also observed in the 500 mg/kg b.wt. aspartame-treated group (Table 1). No significant change in lipid peroxidation was observed in the experimental groups as compared to the control group (Table 2). Table 3 shows the change in various antioxidant enzymes in liver in response to different doses of aspartame after 180 days. No significant change was observed in SOD and catalase activity. GPx and GR activities were significantly decreased in aspartame-treated group (1000 mg/kg b.wt.) where as the changes were insignificant in 500 mg aspartame/kg b.wt. treated group. GSH was also significantly decreased in the experimental groups (Fig. 1). Fig. 2 shows the histopathological changes in the liver of rats. Leukocyte infiltration was observed in liver tissues of 1000 mg/kg b.wt. aspartame administered group whereas the liver of rats administered with 500 mg/kg b.wt. aspartame did not demonstrate any pathological changes. Fig. 3 represents leukocyte infiltration score in liver of the control and experimental groups.

Table 1 Effect of aspartame on serum enzymes. Groups

ALT (IU/L)

AST (IU/L)

ALP (IU/L)

GGT (U/L)

Control Experimental group I (500 mg/ kg b.wt.) Experimental group II (1000 mg/ kg b.wt.)

28.35 ± 1.98 30.73 ± 2.05

21.80 ± 1.58 25.71 ± 1.79b

73.30 ± 1.58 74.40 ± 1.20

6.93 ± 0.99 9.21 ± 0.86c

42.63 ± 2.44a

40.01 ± 1.73a

81.56 ± 2.14a

27.96 ± 1.90a

Data are expressed as mean ± SD, n = 6. a P < 0.001,when compared with control group. b P < 0.01, when compared with control group. c P < 0.05 when compared with control group.

Table 2 Effect of aspartame on lipid peroxidation in the liver of rats (nM of TBARS formed/mg protein). Control 4.20 ± 0.78

Experimental group I (500 mg/kg b.wt.)

Experimental group II (1000 mg/kg b.wt.)

3.98 ± 0.39

4.08 ± 0.35

Data are expressed as mean ± SD, n = 6.

Table 3 Effect of aspartame on activities of antioxidant enzymes in the liver of rats. Antioxidant enzymes

Control

Experimental group I (500 mg/ kg b.wt.)

Experimental group II (1000 mg/ kg b.wt.)

SOD (U/mg protein) Catalase (nkat/mg protein) GPx (lg of GSH consumed/min/ mg protein) GR (nmol of NADPH oxidized/min/mg protein)

9.12 ± 0.31 31.97 ± 2.24

9.10 ± 0.29 31.21 ± 2.08

9.15 ± 0.54 31.05 ± 1.79

11.11 ± 1.12

10.49 ± 0.58

8.01 ± 0.66a

50.39 ± 2.27

48.57 ± 2.87

40.16 ± 4.47a

Data are expressed as mean ± SD, n = 6. a P < 0.001 when compared with control group.

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Fig. 1. Concentration of reduced glutathione (GSH) in aspartame administered rats. Data are expressed as mean ± SD, n = 6. ⁄ Indicates values significantly different from control group (P < 0.05), ⁄⁄ indicates values significantly different from control group (P < 0.001).

4. Discussion The present study highlights the effect of long term consumption of two different doses of aspartame on antioxidant defense system and enzyme markers of hepatocellular injury in the liver. Aspartame is one of the widely consumed artificial sweetener and health conscious societies are increasingly concerned about its safety. Most of the people are unaware about the amount of aspartame they consume through various products. An important and interesting question is whether the chronic uncontrolled consumption of aspartame is safe to humans. The effect of aspartame on human is probably dependent on its metabolite components. We hypothesized that long term consumption of aspartame may cause liver injury which was marked by the increase in AST, ALT, ALP and GGT activities in serum. It is certainly possible that the enhanced activities of these enzymes observed in this study are due to methanol, the byproduct of aspartame metabolism, which is previously reported to produce altered oxidant/antioxidant balance and surface charge density which cause leakage of ALT and AST (Parthasarathy et al., 2006). ALP is a membrane associated enzyme and an increased activity of ALP is an indication of liver damage (Giannini et al., 2005). GGT is a microsomal enzyme present in hepatocytes and its primary role is to metabolize extracellular GSH allowing for precursor amino acids to be assimilated and reutilized for intracellular GSH synthesis. An increase in serum GGT is a defense mechanism reflecting the induction of cellular GGT, when there is oxidative stress (Lee et al., 2004). A small amount of aspartame significantly increases the plasma methanol levels (Davoli, 1986). The antioxidant potential involving cellular GSH content and the activities of related enzymes were decreased in liver during methanol intoxication (Skrzydlewska, 2003). In the present study, activities of GPx and GR of aspartame administered group 1000 mg/kg b.wt. were decreased significantly compared to control group. The decrease in the activities of antioxidant enzymes GPx, GR and GSH may be due to the damaging effect of free radicals produced following methanol exposure or alternatively could be a direct effect of formaldehyde formed from oxidation of methanol, on these enzymes. GPx is involved in the reduction of hydrogen peroxide to water by using glutathione as hydrogen donor (Prabhakar et al., 2005). Diminished activity of GPx results in the enhanced conversion of hydrogen peroxide to hydroxyl radical that may contribute to oxidative stress

Fig. 2. (A) Liver of control rats shows normal architecture with no leukocyte infiltration and necrosis (H&E40). (B) Experimental group I (500 mg/kg b.wt. aspartame) shows normal architecture with no leukocyte infiltration and necrosis (H&E40). (C) Experimental group II (1000 mg/kg b.wt. aspartame) shows leukocyte infiltration (arrow indicates leukocyte infiltration) (H&E40).

(Reiter, 2000). The oxidation of a cysteine SH group adjacent to a selenol group in the active site of GPx is a likely cause of irreversible inactivation of the enzyme in liver. GR plays an important role in cellular antioxidant protection by catalyzing the reduction of GSSG to GSH (Cazenave et al., 2006). GR is the key enzyme in the maintenance of liver GSH. The decrease in GR activity may indicate

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5. Conclusion

Fig. 3. Leukocyte infiltration score in the liver of aspartame administered rats. Data represented as mean ± SD (n = 6). ⁄Indicates values are significantly different from control group. Significance accepted at (P < 0.05).

The present study demonstrated the effects of long term administration of two different doses of aspartame in serum enzymes and antioxidant defense system in liver. Significant increase in the activities of serum enzymes indicates that aspartame may produce liver injury. Glutathione and glutathione dependent enzyme activities were found to have been decreased significantly in group administered with high dose of aspartame. This indicates the toxicity of aspartame is dose dependent. Histopathology of liver showed inflammatory changes. The outcome of the present study is that long term intake of aspartame produces alterations in antioxidant defense status and histopathology in the liver mainly through glutathione dependent system. Further studies are required to evaluate the effect of aspartame in other tissues. Conflict of Interest

a deficit in production of oxidized glutathione back from GSH catalyzed by GPx (Morena et al., 2005). The decrease in activities of these enzymes is probably related with the action of methanol metabolites such as formaldehyde and free radicals. Formaldehyde readily reacts with the amino acids of soluble proteins leading to hydroxymethyl derivatives and intra and intermolecular bridges in proteins. Free radicals formed during the methanol oxidation can also cause formation of protein peroxides. These changes may result in denaturation, aggregation and fragmentation of proteins, altering physicochemical properties and potentially losing of enzymatic activities (Skrzydlewska et al., 2000). Glutathione is an important non-enzymatic antioxidant which plays a critical role in cellular defense system against toxic chemicals of exogenous and endogenous origin. Depletion of cellular GSH increases cell vulnerability to oxidative stress (Oyama et al., 2002). In this study, liver GSH levels of both the experimental groups decreased after 180 days administration, suggesting an imbalance in the prooxidant/antioxidant system. Liver is the major organ of GSH synthesis. The decrease in GSH levels seems to have been caused by methanol intoxication due to aspartame ingestion as methanol metabolism depends on GSH. Glutathione is the cofactor of formaldehyde dehydrogenase and it is responsible for formaldehyde metabolism (Harris et al., 2004). A decrease in glutathione levels reduces formaldehyde metabolism thereby increasing its toxicity. The decrease in GSH may be due to the rapid reaction of GSH with formaldehyde to form nucleophilic adducts (Sogut et al., 2004). The histopathological findings were corroborated by increased activities of serum enzymes and decreased activities of GSH and GSH dependent enzymes. Reactive oxygen species which prevail due to decreased antioxidant defense mechanism may stimulate the release and formation of various inflammatory chemokines. Chemokines are involved in the migration of leukocytes into liver during alcohol intoxication (Bautista, 2002). Chronic intoxication selectively enhances chemokine release by Kupferr cells and hepatic sinusoidal cells and migration of inflammatory cells to liver. Enhanced oxidative stress and reactive oxygen species production following alcohol intoxication may be responsible for the activation of nuclear transcription factor such as NF-jB results in enhanced chemokine production by Kupferr cells (Reinke et al., 2000). The up regulated expression of different members of the chemokine system is likely to result in leukocyte infiltration. Our findings implicate that the effect of aspartame on the antioxidant defense system in the liver is predominant in the glutathione dependent system.

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