- Email: [email protected]
Effect of long intake of aspartame on ionic imbalance in immune organs of immunized wistar albino rats
G Model
ARTICLE IN PRESS
BIOMAG-137; No. of Pages 7
Biomedicine & Aging Pathology xxx (2014) xxx–xxx
Available online at
ScienceDirect www.sciencedirect.com
Original article
Effect of long intake of aspartame on ionic imbalance in immune organs of immunized wistar albino rats Arbind Kumar Choudhary , Sheela Devi Rathinasamy ∗ Department of Physiology, Dr. ALM.PG. Institute of Basic Medical Sciences, University of Madras, Taramani Campus, Chennai, Tamilnadu, India
a r t i c l e
i n f o
Article history: Received 3 March 2014 Accepted 11 March 2014 Available online xxx Keywords: Aspartame ATPase’s LPO Immune organs Immunization
a b s t r a c t Aspartame was approved by the U.S. Food and Drug Administration (FDA) in 1981 for dry food in 1983 for soft drinks and in 1996 for all foods. In 2006, it was approved for use throughout the European Union [EFSA] on safety dose (40 mg/kg b.w). The artificial dipeptide sweetener aspartame [APM; L- aspartyl-Lphenylalanine methyl ester] is present in many products especially unsweetened and sugar products. These products are frequently utilized by people trying to lose weight or patients with diabetes. Concern relating to the possible adverse effect has been raised due to aspartame metabolic components. Aspartame is rapidly and completely metabolized in humans and experimental animals to aspartic acid (40%), phenylalanine (50%) and methanol (10%). Methanol, a toxic metabolite is primarily metabolized by oxidation to formaldehyde and then to formate these processes are accompanied by the formation of superoxide anion and hydrogen peroxide. This study focus is to understand whether the oral administration of aspartame (40 mg/kg b.w.) for 90 days, have any effect on membrane bound ATPase’s, which may cause ionic disproportion and imbalance the homeostasis of immune organs in wistar albino rats. To mimic human methanol metabolism, folate deficient rats were used. After 90 days of aspartame administration, showed a significant alteration in membrane bound ATPase’s and serum ions. Excess free radical generation is confirmed by increase in lipid peroxidation and nitric oxide level. This study concludes that oral administration of aspartame (40 mg/kg b.w.) for longer duration altered the homeostasis of immune organs, may cause oxidative stress in immune organs of wistar albino rats. © 2014 Published by Elsevier Masson SAS.
1. Introduction Aspartame was approved by the U.S. Food and Drug Administration (FDA) in 1981 for dry food [1], in 1983 for soft drinks [2] and in 1996 for all foods [3]. In 1994, it was approved for use throughout the European Union [EFSA, 2006] [4] on safety dose (40 mg/kg b.w). The artificial dipeptide sweetener aspartame [APM; L- aspartyl-L- phenylalanine methylester] is present in many products especially unsweetened and sugar products. These products are frequently utilized by people trying to lose weight or patients with diabetes. Concerns relating to the possible adverse effect have been raised due to aspartame’s metabolic components, which is produced during its breakdown, namely phenylalanine, aspartic acid [aspartate], diketopiperazine [DKP] and methanol [5]. Aspartame can be also absorbed into the mucosal cells prior to hydrolysis and then metabolized within the cell to its three components which then enter circulation [6] (Mattews, in 1984). Methanol is not subject to metabolism within the enterocyte and
∗ Corresponding author. Tel.: +91 98 41 44 70 46; fax: +91 04 424 54 07 09. E-mail address: [email protected] (S.D. Rathinasamy).
rapidly enters the portal circulation and is oxidized in the liver to formaldehyde, a highly reactive chemical which strongly binds to proteins [7] (Haschemeyer and Haschemeyer, in 1973) and nucleic acids [8] (Metzler, in 1977) forming formaldehyde adducts. Membrane ATPase’s may play an important role in ionic gradients between the intracellular/extracellular compartments of the cell. Membrane ATPases are intimately associated with the plasma membrane and participates in the energy requiring translocation of sodium, potassium, calcium and magnesium [9]. Na+ K+ ATPase plays an important role in active transport of Na+ and K+ ions, across the plasma membrane, similarly Ca2+ ATPase is clearly linked with Ca2+ pump and transport of Ca2+ . The Mg2 ions forms Mg2+ ATPase complex, which is the substrate for the enzyme Mg2+ ATPase is to control the intracellular Mg2+ concentration, changes which can modulate the activity of Mg2+ dependent enzymes and regulate rates of protein synthesis and cell growth [10]. Generation of free radicals is an integral feature of normal cellular functions, in contrast, excessive generation and/or inadequate removal of free radical results in destructive and irreversible damage to the cell [11]. Stressor is a stimulus by either internal or external, which activates the hypothalamic pituitary adrenal axis and the sympathetic nervous system resulting in a physiological change.
http://dx.doi.org/10.1016/j.biomag.2014.03.001 2210-5220/© 2014 Published by Elsevier Masson SAS.
Please cite this article in press as: Choudhary AK, Rathinasamy SD. Effect of long intake of aspartame on ionic imbalance in immune organs of immunized wistar albino rats. Biomed Aging Pathol (2014), http://dx.doi.org/10.1016/j.biomag.2014.03.001
G Model BIOMAG-137; No. of Pages 7
ARTICLE IN PRESS A.K. Choudhary, S.D. Rathinasamy / Biomedicine & Aging Pathology xxx (2014) xxx–xxx
2
Corticotrophin-releasing hormone is released during stress and stimulates the release of adrenocorticotropic hormone which in turn releases corticosterone from the adrenal cortex, [12] which indicate that aspartame act as a chemical stressor. The immune system is particularly sensitive to stress and specific effects of stress have been demonstrated by number of studies [13,14], from previous studies on free radical production by exposure of aspartame (40 mg/kg b.w) has been studied in liver and kidney of wistar albino rats [15]. But attention has not been focused on the changes occur in homeostasis of immune organs (like spleen, thymus, lymph node and bone marrow) during the exposure of aspartame. Hence, the focus of the study is to investigate homeostasis of immune organs on exposure of aspartame (40 mg/kg b.w) for longer duration of 90 days. 2. Method 2.1. Animal model Animal experiments were carried out after getting clearance from the Institutional Animal Ethical Committee (IAEC No: 02/03/11) and the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA). The experimental animals were healthy, inbreed adult male Wistar albino rats, weighing approximately 200–220 g (12 weeks of age). The animals were maintained under standard laboratory conditions and were allowed to have food and water ad libitum (standard rat feed pellets supplied by M/s. Hindustan Lever Ltd., India) for animals. Animals of aspartame treated groups were daily-administered aspartame (40 mg/kg b.w) [4] dissolved in normal saline orally (by means of lavarge needle) for 90 days. All the rats were housed under condition of controlled temperature (26 ± 2 ◦ C) with 12 hours light and 12 hours dark exposure. 2.2. Experimental design Group I were the control-immunized animals, which were administered normal saline orally (by means of lavarge needle) thought out the experimental protocol. Group III were controlimmunized animals treated with aspartame orally for 90 days (40 mg/kg b.w). Since human beings have very low hepatic folate content [16]. In methanol, metabolism conversion of formate to carbon dioxide is folate dependent. Hence in the deficiency of folic acid, methanol metablism could take the alternate pathway (microsomal pathway) [17]. To simulate this, rats were made folate deficient by feeding them on a special dietary regime for 37 days and after that methotrexate (MTX) in sterile saline were administered by every other day for two week [18] before euthanasia. MTX folate deficiency was confirmed by estimating the urinary excretion of formaminoglutamic acid (FIGLU) [19] prior to the experiment. Rats on a folate deficient diet excreted an average of 90 mg FIGLU/kg body weight/day (range 25–125) while animals on the control diet excreted an average of 0.29 mg/kg body weight/day (range 0.15–0.55). These folate deficient animals showed a significant increase in FIGLU excretion when compared to the control animals (P < 0.05). The folate deficient animals were further divided into 2 groups. Group II were folate deficient diet fed immunized control, GROUP IV was folate deficient diet fed immunized animals treated with aspartame orally for 90 days (40 mg/kg b.w). All the animals were immunized by giving a single IP dose of 5 × 109 sheep red blood cells (SRBC) on before four days of euthanasia. Experimental groups are: • group I: control immunized animals; • group II: folate deficient immunized control animal;
• group III: control immunized animals treated with aspartame (40 mg/kg b.w) orally for 90 days; • group IV: folate deficient immunized animals treated with aspartame (40 mg/kg b.w) orally for 90 days.
2.3. Sample collection Blood samples and isolation of spleen, thymus, and lymph node was performed between 8 and 10 a.m. to avoid circadian rhythm induced changes. Stress-free blood samples were collected as per the technique described by Feldman and Conforti [20]. At the end of experimental period, all the animals were exposed to mild anesthesia and blood was collected from internal jugular vein, plasma and serum was separated respectively by centrifugation at 3000 r.p.m at 4 ◦ C for 15 minutes. Later, all the animals were sacrificed under deep anesthesia using Pentothal sodium (40 mg/kg b.w). The spleen, thymus and lymph node was excised, washed in ice cold saline and blotted to dryness. Quickly weighed and the spleen, thymus and lymph node sample were homogenized by using Teflon glass homogenizers. Ten percent homogenate of this tissue was prepared in phosphate buffer (0.1 M, pH 7.0) and centrifuged at 3000 g at 4 ◦ C for 15 minutes to remove cell debris and the clear supernatant was used for further biochemical assays.
2.4. Biochemical determinations Estimation of plasma cortisol was determined by the procedure of Clark [21]. The activity of (ATPase) (EC 3.6.1.3) Na+ /K+ ATPase was estimated by the method of Bonting [22]. Ca2+ ATPase by the method of Hjerten and Pan [23] and Mg2+ ATPase by the method of Ohnishi et al., [24] in which the liberated phosphate was estimated according to the method of Fiske and Subbarow [25]. Protein was estimated as per the method described by Lowry et al., [26]. Lipid peroxidation was determined in immune organs as described by Ohkawa et al., [27]. Nitric oxide (NO) levels were measured as total nitrite + nitrate levels with the use of the Griess reagent by the method of Bradford [28]. Organ cell count by cross et al., [29] and electrolytes levels in serum were done by auto analyzer (Beckman Coulter India, Ltd., Mumbai, India).
2.5. Statistical analysis Data are expressed as mean ± standard deviation (SD). All data were analyzed with the SPSS for windows statistical package (version 20.0, SPSS Institute Inc., Cary, North Carolina). Statistical significance between the different groups was determined by one way-analysis of variance (ANOVA). When the groups showed significant difference then Tukey’s multiple comparison tests was followed and the significance level was fixed at P < 0.05.
3. Results 3.1. Effect of aspartame on membrane bound enzymes The data are summarized in (Figs. 1–3) with mean ± SD. The membrane bound enzymes (Na+ K+ ATPase, Mg2+ ATPase and Ca2+ ATPase) in immune organs of folate deficient diet fed animals was similar to control animals. But both the control animals as well as folate deficient diet fed animals treated with aspartame for 90 days, the entire membrane bound enzymes (Na+ K+ ATPase, Mg2+ ATPase and Ca2+ ATPase) was decreased when compared to controls and folate deficient diet fed animals.
Please cite this article in press as: Choudhary AK, Rathinasamy SD. Effect of long intake of aspartame on ionic imbalance in immune organs of immunized wistar albino rats. Biomed Aging Pathol (2014), http://dx.doi.org/10.1016/j.biomag.2014.03.001
G Model
ARTICLE IN PRESS
BIOMAG-137; No. of Pages 7
A.K. Choudhary, S.D. Rathinasamy / Biomedicine & Aging Pathology xxx (2014) xxx–xxx
3
Fig. 1. Effect of aspartame on Na+ K+ ATPase. Each value represents mean ± SD. Significance at *P < 0.05, *a: compared with group I; *b: compared with group II. Group I: immunized control; group II: folate deficient immunized control; group III: immunized control + aspartame; group IV: folate deficient immunized control + aspartame.
Fig. 2. Effect of aspartame on Mg2+ ATPase. Each value represents mean ± SD. Significance at *P < 0.05, *a: compared with group I; *b: compared with group II. Group I: immunized control; group II: folate deficient immunized control; group III: immunized control + aspartame; group IV: folate deficient immunized control + aspartame.
Table 1 Effect of aspartame on serum electrolytes level. Parameters
Group I
Na+ (milli equivt/L) eK+ (milli equivt/L) Ca2+ (mg/dL) Mg2+ (mg/dL)
126.00 4.90 9.00 3.00
Group II ± ± ± ±
1.50 1.30 1.70 0.60
124.00 5.70 10.20 3.30
± ± ± ±
Group III 1.80 1.50 1.55 0.40
87.00 8.60 4.00 1.00
± ± ± ±
Group IV 4.50*a, 1.92*a, 0.80*a, 0.25*a,
*b *b *b *b
91.00 9.00 3.70 0.93
± ± ± ±
6.30*a, 1.78*a, 0.66*a, 0.20*a,
*b *b *b *b
Each value represents mean ± SD. Significance at *P < 0.05, *a: compared with group I; *b: compared with group II. Group I: immunized control; group II: folate deficient immunized control; group III: immunized control + aspartame; group IV: folate deficient immunized control + aspartame.
Please cite this article in press as: Choudhary AK, Rathinasamy SD. Effect of long intake of aspartame on ionic imbalance in immune organs of immunized wistar albino rats. Biomed Aging Pathol (2014), http://dx.doi.org/10.1016/j.biomag.2014.03.001
G Model
ARTICLE IN PRESS
BIOMAG-137; No. of Pages 7
A.K. Choudhary, S.D. Rathinasamy / Biomedicine & Aging Pathology xxx (2014) xxx–xxx
4
Fig. 3. Effect of aspartame on Ca2+ ATPase. Each value represents mean ± SD. Significance at *P < 0.05, *a: compared with group I; *b: compared with group II. Group I: immunized control; group II: folate deficient immunized control; group III: immunized control + aspartame; group IV: folate deficient immunized control + aspartame. Table 2 Effect of aspartame on plasma coticosterone level (g/dL of plasma). Parameters
Group I
Group II
Group III
Group IV
Corticosterone
41.96 ± 1.76
42.58 ± 1.64
93.61 ± 2.55*a, *b
95.44 ± 2.22*a, *b
Each value represents mean ± SD. Significance at *P < 0.05, *a: compared with group I; *b: compared with group II. Group I: immunized control; group II: folate deficient immunized control; group III: immunized control + aspartame; group IV: folate deficient immunized control + aspartame.
3.2. Effect of aspartame on serum electrolyte level
by the elevated corticosteroid level in the aspartame treated group.
The data are presented with mean ± SD; in (Table 1).The electrolytes (Na+ , K+ , Ca2+ & Mg2+ ) in serum of folate deficient diet fed animal was similar to control animals. However, in control and folate deficient diet fed aspartame treated animal for 90 days, potassium level were significantly increased, while sodium, calcium and magnesium levels were significantly decreased in serum when compared to control and folate deficient diet fed control animals.
3.3. Effect of aspartame on plasma coticosterone level The results are summarized in (Table 2) with mean ± SD. The corticosterone level did not get significantly altered in folate deficient diet fed animal when compare to control animal. But both control as well as folate deficient diet fed animal treated with aspartame for 90 days showed significant increase in corticosterone level, when compared to control as well as folate deficient diet fed animal. The present study clearly confirms that aspartame can be act as chemical stressor as indicated
3.4. Effect of aspartame on LPO and nitric oxide level The results are summarized in (Table 3) as mean ± SD. The LPO of folate deficient animals diet fed was similar to the control animals. But both the control animals as well as folate deficient diet fed animals treated with aspartame for 90 days, the LPO level was increased when compared to controls and folate deficient diet fed animals. This clearly indicates the generation of free radicals by aspartame metabolites. The increase level of lipid peroxidation is taken as direct evidence for oxidative stress.
3.5. Effect of aspartame on nitric oxide level The results are summarized in (Table 4) as mean ± SD. The nitric oxide of folate deficient animals diet fed was similar to the control animals. But both the control animals as well as folate deficient diet fed animals treated with aspartame for 90 days, the nitric oxide level was increased when compared to controls and folate deficient
Table 3 Effect of aspartame on lipid peroxidation (LPO) level (moles/mg protein). Organs
Group I
Group II
Group III
Group IV
Spleen Thymus Lymph node
2.79 ± 0.58 3.80 ± 0.62 2.91 ± 0.51
3.19 ± 0.76 4.14 ± 0.84 3.39 ± 0.64
11.90 ± 1.51* * 14.65 ± 1.27*a, *b 12.84 ± 1.15*a, *b a,
b
12.97 ± 1.32*a, *b 15.49 ± 1.55*a, *b 13.72 ± 1.27*a, *b
Each value represents mean ± SD. Significance at *P < 0.05, *a: compared with group I; *b: compared with group II. Group I: immunized control; group II: folate deficient immunized control; group III: immunized control + aspartame; group IV: folate deficient immunized control + aspartame.
Please cite this article in press as: Choudhary AK, Rathinasamy SD. Effect of long intake of aspartame on ionic imbalance in immune organs of immunized wistar albino rats. Biomed Aging Pathol (2014), http://dx.doi.org/10.1016/j.biomag.2014.03.001
G Model
ARTICLE IN PRESS
BIOMAG-137; No. of Pages 7
A.K. Choudhary, S.D. Rathinasamy / Biomedicine & Aging Pathology xxx (2014) xxx–xxx
5
Table 4 Effect of aspartame on nitric oxide (NO) level (moles of nitrite/mg protein). Organs
Group 1
Group 2
Group 3
Group IV
Spleen Thymus Lymph node
9.00 ± 1.23 5.16 ± 0.88 7.11 ± 0.90
10.00 ± 1.42 5.94 ± 0.34 7.84 ± 1.20
19.71 ± 2.54*a, *b 13.07 ± 1.50*a, *b 13.90 ± 1.45*a, *b
21.86 ± 2.35*a, *b 14.12 ± 1.97*a, *b 15.00 ± 1.33*a, *b
Each value represents mean ± SD. Significance at *P < 0.05, *a: compared with group I; *b: compared with group II. Group I: immunized control; group II: folate deficient immunized control; group III: immunized control + aspartame; group IV: folate deficient immunized control + aspartame.
Fig. 4. Effect of aspartame on organ cell count. Each value represents mean ± SD. Significance at *P < 0.05, *a: compared with group I; *b: compared with group II. Group I: immunized control; group II: folate deficient immunized control; group III: immunized control + aspartame; group IV: folate deficient immunized control + aspartame.
diet fed animals. This clearly indicates the generation of free radicals by metabolites of aspartame. 3.6. Effect of aspartame on organ cell count and organ weight The data are presented with mean ± SD, in Figs. 4 and 5. The organ cell count and organ weight of spleen, thymus and lymph node of folate deficient diet fed animal was similar to control animals. But both control as well as folate deficient diet fed animal treated with aspartame for 90 days showed significant decrease in cell count and weight of organs when compared to control as well as folate deficient diet fed animal. 4. Discussion In this study, the folate deficient diet fed animals were used to mimic the human methanol metabolism. However, the folate deficient diet fed animals did not showed any significant changes in the parameters studied and remained similar to controls animals. Generation of free radicals such as peroxyl, alkoxyl and aldehyde radicals can cause severe damage to the membrane bound enzymes such as Ca2+ ATPase, Mg2+ ATPase and Na+ K+ ATPase [30]. Free radicals have been suggested to exert their cytotoxic effect by causing peroxidation of membrane phospholipid [31]. Damage of plasma membrane occurs directly through interaction with membrane component such as the ion dependent ATPase and ion channels and indirectly as a consequence of over cytosolic damage. The decrease in the levels of ATPase’s by the free radicals in the aspartame administered animals could be due to free radical induced cell damage
by methanol metabolite of aspartame and their severe cytotoxic effects, such as increased corticosterone level, lipid peroxidation and nitric oxide in this study. The increase level of lipid peroxidation is taken as direct evidence for oxidative stress [32]. Further high concentration of lipid peroxides has been reported to lead to cytoxicity [33]. LPO in cellular membranes damages polyunsaturated fatty acids tending to reduce membrane fluidity, which is essential for proper functioning of the cell. Hence, the elevated level of LPO in the immune organs after aspartame administration in this study could not be ignored as it could affect the organ functions. This alteration could have been due to the methanol released during aspartame metabolism and the formaldehyde formed during methanol metabolism This is well supported by the report of Parthasarathy et al., [34] who observed an increase LPO level after methanol administration in the lymphoid organs. Similarly, Zararsiz et al., [35] recorded a significant increase in LPO level in the kidney of rats after treatment with formaldehyde. Nitric oxide is thought to react with superoxide anion to gain a radical property, which is also a potent source of oxidative injury [36]. Na+ K+ ATPase uses energy derived from the hydrolysis of ATP to keep a high K+ and a low Na+ concentration in the cytoplasm which in turn provides the driving force for the net movement of other substance such as Ca2+ , amino acids and H+ [37]. Matels et al. [38] showed that lipid peroxidation products can cause DNA damage and directly inhibit protein synthesis including Na+/ K+ ATPase in immune organs of aspartame treated animals. These observations go in parallel with a decrease in Na+ and an increase in K+ levels in the serum of aspartame treated rats The importance of serum ionic Na+ and K+ is correlated with their involvement in many vital
Please cite this article in press as: Choudhary AK, Rathinasamy SD. Effect of long intake of aspartame on ionic imbalance in immune organs of immunized wistar albino rats. Biomed Aging Pathol (2014), http://dx.doi.org/10.1016/j.biomag.2014.03.001
G Model BIOMAG-137; No. of Pages 7 6
ARTICLE IN PRESS A.K. Choudhary, S.D. Rathinasamy / Biomedicine & Aging Pathology xxx (2014) xxx–xxx
Fig. 5. Effect of aspartame on organ weight. Each value represents mean ± SD. Significance at *P < 0.05, *a: compared with group I; *b: compared with group II. Group I: immunized control; group II: folate deficient immunized control; group III: immunized control + aspartame; group IV: folate deficient immunized control + aspartame.
activities of cells and tissues where they are actively transported through cell membranes, beside their role in muscle contraction and nerve impulse conduction. Free intracellular calcium, acting as a second messenger, is crucial for a diverse range of biological functions [39]. Ca2+ ATPase, the enzyme responsible for active calcium transport, is extremely sensitive to hydroperoxides and this may lead to its inhibition. Ca2+ ATPase activity was significantly lowered by methanol metabolite of aspartame in the aspartame treated animals. Further Ca2+ ATPase activity is mainly impaired due to oxidative modification of thiol groups present in this enzyme which in turn is due to the generation of free radicals [40]. This could be the reason for decrease in serum Ca2+ level. Mg2+ ATPase is to control the intracellular Mg2+ concentration which can modulate the activity of Mg2+ dependent enzymes and regulate rates of protein synthesis and cell growth [41]. Mg2+ ATPase activity was significantly lowered in the immune organs of aspartame treated animals. This could be the reason in decrease of serum Mg2+ level. Serum magnesium is known to be act as a cofactor for the activation of Na+ K+ ATPase [42,43]. Hence, in this observation, decreased serum Mg2+ level could also be the reason for inhibition of Na+ K+ ATPase activity. The decrease in the levels of ATPases by the free radicals in the aspartame administered animals could be due to free radical induced cell damage by methanol metabolite of aspartame and their severe cytotoxic effects, such as lipid peroxidation in cell membrane followed by the alteration of the membrane fluidity, enzyme properties and ion transport. Enhanced lipid peroxidation which may act on the sulphhydryl groups present in the active sites of the ATPases [44]. Since the membrane bound enzymes are ‘SH’ group containing enzymes [45] and these enzymes are extremely sensitive to hydroperoxides and superoxide radicals [46]. Thiol modification (i.e. loss of protein sulfhydryl group) has been recognized as a critical event in cytotoxicity [47]. The present study clearly confirms that aspartame can be act as chemical stressor as indicated by the elevated corticosteroid level in the aspartame treated group. Increased corticosterone has been shown to decrease the size and weight of the spleen and thymus. The significant reduction in organ weight and organ cell count may
be due to oxidative damage which is studied by Skrzydlewksa and Szynaka [48] who reported that oxidative damage caused marked organ weight loss in albino rats upon methanol intoxication (this is also reported by Parthasarathy et al., [34]). Formaldehyde, the first metabolite of methanol increases the population of shrunken cells, dead cells and hydolipid cells [49] which might be the reason for decreased cellularity (reduction in organ weight and cell count) as with our observation. 5. Conclusion The results of present study clearly point out that aspartame induce oxidative stress by generation of free radicals. Such induced oxidative stress result in inhibiting function of ion-dependent Atpase’s leads to disruption in ion homeostasis, resulting altered cellular metabolism, change in cell membrane fluidity and disturbance of vital function of immune organs which also decreased the cellularity (reduction in organ weight and cell count) of these organs. Aspartame metabolite methanol or formaldehyde may be the causative factors behind the changes observed. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgments The authors gratefully acknowledge the University of Madras for their financial support (UGC No.D.1. (C)/TE/2012/1868). The authors acknowledge Mr. Sunderaswaran loganathan & Mr. Annadurai for their constant support and help. References [1] Food Drug Administration (FDA). Aspartame: Commissioner’s final decision. Fed Reg 1981;46:38285–308. [2] Food Drug Administration (FDA). Food additives permitted for direct addition to food for human consumption: Aspartame. Fed Reg 1983;48:31376–82.
Please cite this article in press as: Choudhary AK, Rathinasamy SD. Effect of long intake of aspartame on ionic imbalance in immune organs of immunized wistar albino rats. Biomed Aging Pathol (2014), http://dx.doi.org/10.1016/j.biomag.2014.03.001
G Model BIOMAG-137; No. of Pages 7
ARTICLE IN PRESS A.K. Choudhary, S.D. Rathinasamy / Biomedicine & Aging Pathology xxx (2014) xxx–xxx
[3] Food Drug Administration (FDA). Food additives permitted for direct addition to food for human consumption: aspartame. Fed Reg 1996;61: 33654–6. [4] European Food Safety Authority (EFSA). Opinion of the Scientific Panel on food additives, flavourings, processing aids and materials in contact with food (AFC) on a request from the Commission related to a new long-term carcinogenicity study on aspartame. Question number EFSA-Q-2005-122. Adopted on 3 May 2006. EFSA J 2006;356:1–44. [5] Trocho C, Pardo R, Rafecas I, Virgili J, Remesar X, Fernandez-Lopez JA, et al. Formaldehyde derived from dietary aspartame binds to tissue components in vivo. Life Sci 1998;63:337–49. [6] Mattews DM. Absorption of peptides, amino acids, and their methylated derivatives. In: Stegink LD, Filer Jr LJ, editors. Aspartame physiology and biochemistry. New York: Dekker; 1984. p. 29–46. [7] Haschemeyer RH, Haschemeyer AEV. Proteins. New York: John Wiley & Sons; 1973. p. 11–30. [8] Metzler DE. Biochemistry. New York: Academic Press; 1977. p. 90–107. [9] Kosawer NJ, Zipser Y, Faltin Z. Membrane thiol disulfide status in glucose6-phosphate dehydrogenase deficient red cells. Relationship to cellular glutathione. Biochim Biophys Acta 1991;1058:152–60. [10] Stekhoren MA. Bonting SL. Transport ATPases: properties and functions. Physiol Rev 1981;61:1–76. [11] Lopaczyski W, Zeisel SH. Antioxidants, programmed cell death, and cancer. Nutr Res 2001;21:295–307. [12] McIntosh LJ, Sapolsky RM. Glucocorticoids increase the accumulation of reactive oxygen species and enhance adriamycin-induced toxicity in neuronal culture. Exp Neurol 1996;141:201–6. [13] McEwen BS. The neurobiology of stress: from serendipity to clinical relevance (1). Brain Res 2000;886:172–89. [14] Mastorakos G, Ilias I. Maternal hypothalamic-pituitary-adrenal axis in pregnancy and the postpartum period, Postpartumrelated disorders. Ann N Y Acad Sci 2000;900:95–106. [15] Mourad M. Effect of aspartame on some oxidative stress parameters in liver and kidney of rats. AJPP 2011;5(6):672–8. [16] Maker AB, Tephly TR. Methanol in poisoning folate deficient rats. Nature 1976;261:715–6. [17] Tephly TR. The toxicity of methanol. Life Sciences 1991;48:1031–41. [18] Ming H, Wang SY, Craig A, Meadows Shirley W. Thenen methotrexate effects on folate status and the deoxyuridine suppression test in rats. Nut Re 1989;9:431–44. [19] Rabinowitz JC, Pricer WE. Formiminotetrahydrofolic acid and methenyltetrahydrofolic acid as intermediates in the formation of N10-formyltetrahydrofolic acid. J Am Chem Soc 1956;78(21):5702–4. [20] Feldman S, Conforti N. Participation of dorsal hippocampus in the glucocorticoids feedback effect on adrenocortical activity. Neuroendocrinology 1980;30:52–5. [21] Clark I. A colometric reaction for the estimation of cortisone, hrdrocortisone, aldosterone and related steroids. Nature 1955;75:123–4. [22] Bonting SL. Sodium potassium activated adenosine triphosphatase and cation transport. In: Bitler EE, editor. Membrane and ion transport, 1. London: Interscience Wiley; 1970. p. 257–63. [23] Hjerten S, Pan H. Purification and characterization of two forms of a low affinity Ca2+ ATPase from erythrocyte membranes. Biochim Biophys Acta 1983;728:281–8. [24] Ohnishi T, Suzuki T, Suzuki Y, Ozawa K. A comparative study of plasma membrane Mg2+ ATPase activities in normal, regenerating and malignant cells. Biochim Biophys Acta 1982;684:67–74. [25] Fiske CH, Subbarow Y. The colorimetric determination of phosphorous. J Biol Chem 1925;663:75–400.
7
[26] Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265–75. [27] Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 1979;95:351–8. [28] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248–54. [29] Cross RJ, Brooks WH, Roszman TL. Hypothalamic immune interaction. J NeurolSci 1982;53:557–66. [30] Pragasam V, Kalaiselvi P, Sumitra K, et al. Counterction of oxalate induced nitrosative stress by supplementation of L- arginine, a potent antilithic agent. Clin Chim Acta 2005;354:159–66. [31] Gubdjorson S, Hallgrimson J, Skuldottir G. Arterial pollution. In: Peter H, Geshaw GA, Paoethi, editors. Properties of transport adenosine triphosphatase. New York: Plenum Publishing Corp; 1983. p. 101. [32] Stefano GB, Fricchione GL, Slingsby BT, Benson H. The placebo effect and relaxation response: neural processes and their coupling to constitutive nitric oxide. Brain Res Brain Res Rev 2001;35(1):1–19. [33] Weglicki WB, Dickens BF, Mak IT. Enhanced lysosomal phospholipid degradation and lysphospholipid production due to free radical. Biochem Biophys 1984;124:229–35. [34] Parthasarathy JN, Ramasundaram SK, Sundaramahalingam M, et al. Methanol induced oxidative stress in ratlymphoid organs. J Occup Health 2006;48:20–7. [35] Zararsiz I, Sarsilmaz M, Tas U, et al. Protective effect of melatonin against formaldehyde-induced kidney damage in rats. Toxicol Ind Health 2007;23(10):573–9. [36] Jaeschke H. The role of reactive oxygen species in hepatic ischaemiareperfusion injury and preconditioning. J Inv Surg 2003;16:127–40. [37] Contreras RG, Shoshani L, Flores-Maldonado CA, et al. Relationship between Na+K+ Atpase and cell attachment. J Cell Sci 1999;112:4223–32. [38] Matels JM, Gomes CP, De Castro IN. Antioxidant enzymes and human disease. Biochem Pharmacol 1999;28:251–3. [39] Berridge MJ, Lipp P, Bootman MD. The versatility and universality of calcium signaling. Nat Rev Mol Cell Biol 2000;1:11–21. [40] Jain KS, Shohet SB. Calcium potentiates the peroxidation of erythrocytemembrane lipids. Biochem Biophys Acta 1981;642:46–54. [41] Sanu H, Rubin H. Ions, cell proliferation and cancer. In: The role of magenisum in cell proliferation and transformation. New York: Academic press; 1982. p. 517–37. [42] Ebel H, Gunther T. Role of magnesium in cardiac diseases. J Clin Biochem 1983;21:249. [43] Altura BM, Altura BT. New perspectives on the role of magnesium in the pathophysiology of cardiovascular system. Clinical aspects. Magnesium 1985;4:226. [44] Pragasam S, Jenitha V, Kalaiselvi X, et al. Oxidative stress a predisposing factor for renal stone formation – amelioration by vitamin E supplementation. Clin Chem Acta 2004;350:57–63. [45] Upasani CD, Balaraman R. Effect of vitamin E, vitamin C and spirulina on the levels of membrane bound enzymes and lipids in some organs of rats exposed to lead. Ind J Pharmacol 2001;33:185–91. [46] Jain KS, Shohet SB. Calcium potentiates the peroxidation of erythrocyte membrane lipids. Biochem Biophys Acta 1981;642:46–54. [47] Pasoe GA, Olafsdottir K, Reed DJ. Vitamin E protection against chemical induced injury. I. Maintenance of cellular protein thiols as a cytoprotective mechanism. Arch Biochem Biophy 1987;256:150–68. [48] Skrzydlewksa E, Szynaka B. Ultrastructral elevation of lysosome and biochemical change in cathepsin D distribution in hepatocytes in methanol intoxication. Roczniki Akademii Medycznej W Bialymstoku 1997;42:47–55. [49] Nakao H, Umebayashi C, Nakata M, Nishizaki Y, Noda K, Okanao Y, et al. Formal dehyde induced shrinkage of rat thymocyte. J Pharmacol Sci 2003;91:83–6.
Please cite this article in press as: Choudhary AK, Rathinasamy SD. Effect of long intake of aspartame on ionic imbalance in immune organs of immunized wistar albino rats. Biomed Aging Pathol (2014), http://dx.doi.org/10.1016/j.biomag.2014.03.001
ARTICLE IN PRESS
BIOMAG-137; No. of Pages 7
Biomedicine & Aging Pathology xxx (2014) xxx–xxx
Available online at
ScienceDirect www.sciencedirect.com
Original article
Effect of long intake of aspartame on ionic imbalance in immune organs of immunized wistar albino rats Arbind Kumar Choudhary , Sheela Devi Rathinasamy ∗ Department of Physiology, Dr. ALM.PG. Institute of Basic Medical Sciences, University of Madras, Taramani Campus, Chennai, Tamilnadu, India
a r t i c l e
i n f o
Article history: Received 3 March 2014 Accepted 11 March 2014 Available online xxx Keywords: Aspartame ATPase’s LPO Immune organs Immunization
a b s t r a c t Aspartame was approved by the U.S. Food and Drug Administration (FDA) in 1981 for dry food in 1983 for soft drinks and in 1996 for all foods. In 2006, it was approved for use throughout the European Union [EFSA] on safety dose (40 mg/kg b.w). The artificial dipeptide sweetener aspartame [APM; L- aspartyl-Lphenylalanine methyl ester] is present in many products especially unsweetened and sugar products. These products are frequently utilized by people trying to lose weight or patients with diabetes. Concern relating to the possible adverse effect has been raised due to aspartame metabolic components. Aspartame is rapidly and completely metabolized in humans and experimental animals to aspartic acid (40%), phenylalanine (50%) and methanol (10%). Methanol, a toxic metabolite is primarily metabolized by oxidation to formaldehyde and then to formate these processes are accompanied by the formation of superoxide anion and hydrogen peroxide. This study focus is to understand whether the oral administration of aspartame (40 mg/kg b.w.) for 90 days, have any effect on membrane bound ATPase’s, which may cause ionic disproportion and imbalance the homeostasis of immune organs in wistar albino rats. To mimic human methanol metabolism, folate deficient rats were used. After 90 days of aspartame administration, showed a significant alteration in membrane bound ATPase’s and serum ions. Excess free radical generation is confirmed by increase in lipid peroxidation and nitric oxide level. This study concludes that oral administration of aspartame (40 mg/kg b.w.) for longer duration altered the homeostasis of immune organs, may cause oxidative stress in immune organs of wistar albino rats. © 2014 Published by Elsevier Masson SAS.
1. Introduction Aspartame was approved by the U.S. Food and Drug Administration (FDA) in 1981 for dry food [1], in 1983 for soft drinks [2] and in 1996 for all foods [3]. In 1994, it was approved for use throughout the European Union [EFSA, 2006] [4] on safety dose (40 mg/kg b.w). The artificial dipeptide sweetener aspartame [APM; L- aspartyl-L- phenylalanine methylester] is present in many products especially unsweetened and sugar products. These products are frequently utilized by people trying to lose weight or patients with diabetes. Concerns relating to the possible adverse effect have been raised due to aspartame’s metabolic components, which is produced during its breakdown, namely phenylalanine, aspartic acid [aspartate], diketopiperazine [DKP] and methanol [5]. Aspartame can be also absorbed into the mucosal cells prior to hydrolysis and then metabolized within the cell to its three components which then enter circulation [6] (Mattews, in 1984). Methanol is not subject to metabolism within the enterocyte and
∗ Corresponding author. Tel.: +91 98 41 44 70 46; fax: +91 04 424 54 07 09. E-mail address: [email protected] (S.D. Rathinasamy).
rapidly enters the portal circulation and is oxidized in the liver to formaldehyde, a highly reactive chemical which strongly binds to proteins [7] (Haschemeyer and Haschemeyer, in 1973) and nucleic acids [8] (Metzler, in 1977) forming formaldehyde adducts. Membrane ATPase’s may play an important role in ionic gradients between the intracellular/extracellular compartments of the cell. Membrane ATPases are intimately associated with the plasma membrane and participates in the energy requiring translocation of sodium, potassium, calcium and magnesium [9]. Na+ K+ ATPase plays an important role in active transport of Na+ and K+ ions, across the plasma membrane, similarly Ca2+ ATPase is clearly linked with Ca2+ pump and transport of Ca2+ . The Mg2 ions forms Mg2+ ATPase complex, which is the substrate for the enzyme Mg2+ ATPase is to control the intracellular Mg2+ concentration, changes which can modulate the activity of Mg2+ dependent enzymes and regulate rates of protein synthesis and cell growth [10]. Generation of free radicals is an integral feature of normal cellular functions, in contrast, excessive generation and/or inadequate removal of free radical results in destructive and irreversible damage to the cell [11]. Stressor is a stimulus by either internal or external, which activates the hypothalamic pituitary adrenal axis and the sympathetic nervous system resulting in a physiological change.
http://dx.doi.org/10.1016/j.biomag.2014.03.001 2210-5220/© 2014 Published by Elsevier Masson SAS.
Please cite this article in press as: Choudhary AK, Rathinasamy SD. Effect of long intake of aspartame on ionic imbalance in immune organs of immunized wistar albino rats. Biomed Aging Pathol (2014), http://dx.doi.org/10.1016/j.biomag.2014.03.001
G Model BIOMAG-137; No. of Pages 7
ARTICLE IN PRESS A.K. Choudhary, S.D. Rathinasamy / Biomedicine & Aging Pathology xxx (2014) xxx–xxx
2
Corticotrophin-releasing hormone is released during stress and stimulates the release of adrenocorticotropic hormone which in turn releases corticosterone from the adrenal cortex, [12] which indicate that aspartame act as a chemical stressor. The immune system is particularly sensitive to stress and specific effects of stress have been demonstrated by number of studies [13,14], from previous studies on free radical production by exposure of aspartame (40 mg/kg b.w) has been studied in liver and kidney of wistar albino rats [15]. But attention has not been focused on the changes occur in homeostasis of immune organs (like spleen, thymus, lymph node and bone marrow) during the exposure of aspartame. Hence, the focus of the study is to investigate homeostasis of immune organs on exposure of aspartame (40 mg/kg b.w) for longer duration of 90 days. 2. Method 2.1. Animal model Animal experiments were carried out after getting clearance from the Institutional Animal Ethical Committee (IAEC No: 02/03/11) and the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA). The experimental animals were healthy, inbreed adult male Wistar albino rats, weighing approximately 200–220 g (12 weeks of age). The animals were maintained under standard laboratory conditions and were allowed to have food and water ad libitum (standard rat feed pellets supplied by M/s. Hindustan Lever Ltd., India) for animals. Animals of aspartame treated groups were daily-administered aspartame (40 mg/kg b.w) [4] dissolved in normal saline orally (by means of lavarge needle) for 90 days. All the rats were housed under condition of controlled temperature (26 ± 2 ◦ C) with 12 hours light and 12 hours dark exposure. 2.2. Experimental design Group I were the control-immunized animals, which were administered normal saline orally (by means of lavarge needle) thought out the experimental protocol. Group III were controlimmunized animals treated with aspartame orally for 90 days (40 mg/kg b.w). Since human beings have very low hepatic folate content [16]. In methanol, metabolism conversion of formate to carbon dioxide is folate dependent. Hence in the deficiency of folic acid, methanol metablism could take the alternate pathway (microsomal pathway) [17]. To simulate this, rats were made folate deficient by feeding them on a special dietary regime for 37 days and after that methotrexate (MTX) in sterile saline were administered by every other day for two week [18] before euthanasia. MTX folate deficiency was confirmed by estimating the urinary excretion of formaminoglutamic acid (FIGLU) [19] prior to the experiment. Rats on a folate deficient diet excreted an average of 90 mg FIGLU/kg body weight/day (range 25–125) while animals on the control diet excreted an average of 0.29 mg/kg body weight/day (range 0.15–0.55). These folate deficient animals showed a significant increase in FIGLU excretion when compared to the control animals (P < 0.05). The folate deficient animals were further divided into 2 groups. Group II were folate deficient diet fed immunized control, GROUP IV was folate deficient diet fed immunized animals treated with aspartame orally for 90 days (40 mg/kg b.w). All the animals were immunized by giving a single IP dose of 5 × 109 sheep red blood cells (SRBC) on before four days of euthanasia. Experimental groups are: • group I: control immunized animals; • group II: folate deficient immunized control animal;
• group III: control immunized animals treated with aspartame (40 mg/kg b.w) orally for 90 days; • group IV: folate deficient immunized animals treated with aspartame (40 mg/kg b.w) orally for 90 days.
2.3. Sample collection Blood samples and isolation of spleen, thymus, and lymph node was performed between 8 and 10 a.m. to avoid circadian rhythm induced changes. Stress-free blood samples were collected as per the technique described by Feldman and Conforti [20]. At the end of experimental period, all the animals were exposed to mild anesthesia and blood was collected from internal jugular vein, plasma and serum was separated respectively by centrifugation at 3000 r.p.m at 4 ◦ C for 15 minutes. Later, all the animals were sacrificed under deep anesthesia using Pentothal sodium (40 mg/kg b.w). The spleen, thymus and lymph node was excised, washed in ice cold saline and blotted to dryness. Quickly weighed and the spleen, thymus and lymph node sample were homogenized by using Teflon glass homogenizers. Ten percent homogenate of this tissue was prepared in phosphate buffer (0.1 M, pH 7.0) and centrifuged at 3000 g at 4 ◦ C for 15 minutes to remove cell debris and the clear supernatant was used for further biochemical assays.
2.4. Biochemical determinations Estimation of plasma cortisol was determined by the procedure of Clark [21]. The activity of (ATPase) (EC 3.6.1.3) Na+ /K+ ATPase was estimated by the method of Bonting [22]. Ca2+ ATPase by the method of Hjerten and Pan [23] and Mg2+ ATPase by the method of Ohnishi et al., [24] in which the liberated phosphate was estimated according to the method of Fiske and Subbarow [25]. Protein was estimated as per the method described by Lowry et al., [26]. Lipid peroxidation was determined in immune organs as described by Ohkawa et al., [27]. Nitric oxide (NO) levels were measured as total nitrite + nitrate levels with the use of the Griess reagent by the method of Bradford [28]. Organ cell count by cross et al., [29] and electrolytes levels in serum were done by auto analyzer (Beckman Coulter India, Ltd., Mumbai, India).
2.5. Statistical analysis Data are expressed as mean ± standard deviation (SD). All data were analyzed with the SPSS for windows statistical package (version 20.0, SPSS Institute Inc., Cary, North Carolina). Statistical significance between the different groups was determined by one way-analysis of variance (ANOVA). When the groups showed significant difference then Tukey’s multiple comparison tests was followed and the significance level was fixed at P < 0.05.
3. Results 3.1. Effect of aspartame on membrane bound enzymes The data are summarized in (Figs. 1–3) with mean ± SD. The membrane bound enzymes (Na+ K+ ATPase, Mg2+ ATPase and Ca2+ ATPase) in immune organs of folate deficient diet fed animals was similar to control animals. But both the control animals as well as folate deficient diet fed animals treated with aspartame for 90 days, the entire membrane bound enzymes (Na+ K+ ATPase, Mg2+ ATPase and Ca2+ ATPase) was decreased when compared to controls and folate deficient diet fed animals.
Please cite this article in press as: Choudhary AK, Rathinasamy SD. Effect of long intake of aspartame on ionic imbalance in immune organs of immunized wistar albino rats. Biomed Aging Pathol (2014), http://dx.doi.org/10.1016/j.biomag.2014.03.001
G Model
ARTICLE IN PRESS
BIOMAG-137; No. of Pages 7
A.K. Choudhary, S.D. Rathinasamy / Biomedicine & Aging Pathology xxx (2014) xxx–xxx
3
Fig. 1. Effect of aspartame on Na+ K+ ATPase. Each value represents mean ± SD. Significance at *P < 0.05, *a: compared with group I; *b: compared with group II. Group I: immunized control; group II: folate deficient immunized control; group III: immunized control + aspartame; group IV: folate deficient immunized control + aspartame.
Fig. 2. Effect of aspartame on Mg2+ ATPase. Each value represents mean ± SD. Significance at *P < 0.05, *a: compared with group I; *b: compared with group II. Group I: immunized control; group II: folate deficient immunized control; group III: immunized control + aspartame; group IV: folate deficient immunized control + aspartame.
Table 1 Effect of aspartame on serum electrolytes level. Parameters
Group I
Na+ (milli equivt/L) eK+ (milli equivt/L) Ca2+ (mg/dL) Mg2+ (mg/dL)
126.00 4.90 9.00 3.00
Group II ± ± ± ±
1.50 1.30 1.70 0.60
124.00 5.70 10.20 3.30
± ± ± ±
Group III 1.80 1.50 1.55 0.40
87.00 8.60 4.00 1.00
± ± ± ±
Group IV 4.50*a, 1.92*a, 0.80*a, 0.25*a,
*b *b *b *b
91.00 9.00 3.70 0.93
± ± ± ±
6.30*a, 1.78*a, 0.66*a, 0.20*a,
*b *b *b *b
Each value represents mean ± SD. Significance at *P < 0.05, *a: compared with group I; *b: compared with group II. Group I: immunized control; group II: folate deficient immunized control; group III: immunized control + aspartame; group IV: folate deficient immunized control + aspartame.
Please cite this article in press as: Choudhary AK, Rathinasamy SD. Effect of long intake of aspartame on ionic imbalance in immune organs of immunized wistar albino rats. Biomed Aging Pathol (2014), http://dx.doi.org/10.1016/j.biomag.2014.03.001
G Model
ARTICLE IN PRESS
BIOMAG-137; No. of Pages 7
A.K. Choudhary, S.D. Rathinasamy / Biomedicine & Aging Pathology xxx (2014) xxx–xxx
4
Fig. 3. Effect of aspartame on Ca2+ ATPase. Each value represents mean ± SD. Significance at *P < 0.05, *a: compared with group I; *b: compared with group II. Group I: immunized control; group II: folate deficient immunized control; group III: immunized control + aspartame; group IV: folate deficient immunized control + aspartame. Table 2 Effect of aspartame on plasma coticosterone level (g/dL of plasma). Parameters
Group I
Group II
Group III
Group IV
Corticosterone
41.96 ± 1.76
42.58 ± 1.64
93.61 ± 2.55*a, *b
95.44 ± 2.22*a, *b
Each value represents mean ± SD. Significance at *P < 0.05, *a: compared with group I; *b: compared with group II. Group I: immunized control; group II: folate deficient immunized control; group III: immunized control + aspartame; group IV: folate deficient immunized control + aspartame.
3.2. Effect of aspartame on serum electrolyte level
by the elevated corticosteroid level in the aspartame treated group.
The data are presented with mean ± SD; in (Table 1).The electrolytes (Na+ , K+ , Ca2+ & Mg2+ ) in serum of folate deficient diet fed animal was similar to control animals. However, in control and folate deficient diet fed aspartame treated animal for 90 days, potassium level were significantly increased, while sodium, calcium and magnesium levels were significantly decreased in serum when compared to control and folate deficient diet fed control animals.
3.3. Effect of aspartame on plasma coticosterone level The results are summarized in (Table 2) with mean ± SD. The corticosterone level did not get significantly altered in folate deficient diet fed animal when compare to control animal. But both control as well as folate deficient diet fed animal treated with aspartame for 90 days showed significant increase in corticosterone level, when compared to control as well as folate deficient diet fed animal. The present study clearly confirms that aspartame can be act as chemical stressor as indicated
3.4. Effect of aspartame on LPO and nitric oxide level The results are summarized in (Table 3) as mean ± SD. The LPO of folate deficient animals diet fed was similar to the control animals. But both the control animals as well as folate deficient diet fed animals treated with aspartame for 90 days, the LPO level was increased when compared to controls and folate deficient diet fed animals. This clearly indicates the generation of free radicals by aspartame metabolites. The increase level of lipid peroxidation is taken as direct evidence for oxidative stress.
3.5. Effect of aspartame on nitric oxide level The results are summarized in (Table 4) as mean ± SD. The nitric oxide of folate deficient animals diet fed was similar to the control animals. But both the control animals as well as folate deficient diet fed animals treated with aspartame for 90 days, the nitric oxide level was increased when compared to controls and folate deficient
Table 3 Effect of aspartame on lipid peroxidation (LPO) level (moles/mg protein). Organs
Group I
Group II
Group III
Group IV
Spleen Thymus Lymph node
2.79 ± 0.58 3.80 ± 0.62 2.91 ± 0.51
3.19 ± 0.76 4.14 ± 0.84 3.39 ± 0.64
11.90 ± 1.51* * 14.65 ± 1.27*a, *b 12.84 ± 1.15*a, *b a,
b
12.97 ± 1.32*a, *b 15.49 ± 1.55*a, *b 13.72 ± 1.27*a, *b
Each value represents mean ± SD. Significance at *P < 0.05, *a: compared with group I; *b: compared with group II. Group I: immunized control; group II: folate deficient immunized control; group III: immunized control + aspartame; group IV: folate deficient immunized control + aspartame.
Please cite this article in press as: Choudhary AK, Rathinasamy SD. Effect of long intake of aspartame on ionic imbalance in immune organs of immunized wistar albino rats. Biomed Aging Pathol (2014), http://dx.doi.org/10.1016/j.biomag.2014.03.001
G Model
ARTICLE IN PRESS
BIOMAG-137; No. of Pages 7
A.K. Choudhary, S.D. Rathinasamy / Biomedicine & Aging Pathology xxx (2014) xxx–xxx
5
Table 4 Effect of aspartame on nitric oxide (NO) level (moles of nitrite/mg protein). Organs
Group 1
Group 2
Group 3
Group IV
Spleen Thymus Lymph node
9.00 ± 1.23 5.16 ± 0.88 7.11 ± 0.90
10.00 ± 1.42 5.94 ± 0.34 7.84 ± 1.20
19.71 ± 2.54*a, *b 13.07 ± 1.50*a, *b 13.90 ± 1.45*a, *b
21.86 ± 2.35*a, *b 14.12 ± 1.97*a, *b 15.00 ± 1.33*a, *b
Each value represents mean ± SD. Significance at *P < 0.05, *a: compared with group I; *b: compared with group II. Group I: immunized control; group II: folate deficient immunized control; group III: immunized control + aspartame; group IV: folate deficient immunized control + aspartame.
Fig. 4. Effect of aspartame on organ cell count. Each value represents mean ± SD. Significance at *P < 0.05, *a: compared with group I; *b: compared with group II. Group I: immunized control; group II: folate deficient immunized control; group III: immunized control + aspartame; group IV: folate deficient immunized control + aspartame.
diet fed animals. This clearly indicates the generation of free radicals by metabolites of aspartame. 3.6. Effect of aspartame on organ cell count and organ weight The data are presented with mean ± SD, in Figs. 4 and 5. The organ cell count and organ weight of spleen, thymus and lymph node of folate deficient diet fed animal was similar to control animals. But both control as well as folate deficient diet fed animal treated with aspartame for 90 days showed significant decrease in cell count and weight of organs when compared to control as well as folate deficient diet fed animal. 4. Discussion In this study, the folate deficient diet fed animals were used to mimic the human methanol metabolism. However, the folate deficient diet fed animals did not showed any significant changes in the parameters studied and remained similar to controls animals. Generation of free radicals such as peroxyl, alkoxyl and aldehyde radicals can cause severe damage to the membrane bound enzymes such as Ca2+ ATPase, Mg2+ ATPase and Na+ K+ ATPase [30]. Free radicals have been suggested to exert their cytotoxic effect by causing peroxidation of membrane phospholipid [31]. Damage of plasma membrane occurs directly through interaction with membrane component such as the ion dependent ATPase and ion channels and indirectly as a consequence of over cytosolic damage. The decrease in the levels of ATPase’s by the free radicals in the aspartame administered animals could be due to free radical induced cell damage
by methanol metabolite of aspartame and their severe cytotoxic effects, such as increased corticosterone level, lipid peroxidation and nitric oxide in this study. The increase level of lipid peroxidation is taken as direct evidence for oxidative stress [32]. Further high concentration of lipid peroxides has been reported to lead to cytoxicity [33]. LPO in cellular membranes damages polyunsaturated fatty acids tending to reduce membrane fluidity, which is essential for proper functioning of the cell. Hence, the elevated level of LPO in the immune organs after aspartame administration in this study could not be ignored as it could affect the organ functions. This alteration could have been due to the methanol released during aspartame metabolism and the formaldehyde formed during methanol metabolism This is well supported by the report of Parthasarathy et al., [34] who observed an increase LPO level after methanol administration in the lymphoid organs. Similarly, Zararsiz et al., [35] recorded a significant increase in LPO level in the kidney of rats after treatment with formaldehyde. Nitric oxide is thought to react with superoxide anion to gain a radical property, which is also a potent source of oxidative injury [36]. Na+ K+ ATPase uses energy derived from the hydrolysis of ATP to keep a high K+ and a low Na+ concentration in the cytoplasm which in turn provides the driving force for the net movement of other substance such as Ca2+ , amino acids and H+ [37]. Matels et al. [38] showed that lipid peroxidation products can cause DNA damage and directly inhibit protein synthesis including Na+/ K+ ATPase in immune organs of aspartame treated animals. These observations go in parallel with a decrease in Na+ and an increase in K+ levels in the serum of aspartame treated rats The importance of serum ionic Na+ and K+ is correlated with their involvement in many vital
Please cite this article in press as: Choudhary AK, Rathinasamy SD. Effect of long intake of aspartame on ionic imbalance in immune organs of immunized wistar albino rats. Biomed Aging Pathol (2014), http://dx.doi.org/10.1016/j.biomag.2014.03.001
G Model BIOMAG-137; No. of Pages 7 6
ARTICLE IN PRESS A.K. Choudhary, S.D. Rathinasamy / Biomedicine & Aging Pathology xxx (2014) xxx–xxx
Fig. 5. Effect of aspartame on organ weight. Each value represents mean ± SD. Significance at *P < 0.05, *a: compared with group I; *b: compared with group II. Group I: immunized control; group II: folate deficient immunized control; group III: immunized control + aspartame; group IV: folate deficient immunized control + aspartame.
activities of cells and tissues where they are actively transported through cell membranes, beside their role in muscle contraction and nerve impulse conduction. Free intracellular calcium, acting as a second messenger, is crucial for a diverse range of biological functions [39]. Ca2+ ATPase, the enzyme responsible for active calcium transport, is extremely sensitive to hydroperoxides and this may lead to its inhibition. Ca2+ ATPase activity was significantly lowered by methanol metabolite of aspartame in the aspartame treated animals. Further Ca2+ ATPase activity is mainly impaired due to oxidative modification of thiol groups present in this enzyme which in turn is due to the generation of free radicals [40]. This could be the reason for decrease in serum Ca2+ level. Mg2+ ATPase is to control the intracellular Mg2+ concentration which can modulate the activity of Mg2+ dependent enzymes and regulate rates of protein synthesis and cell growth [41]. Mg2+ ATPase activity was significantly lowered in the immune organs of aspartame treated animals. This could be the reason in decrease of serum Mg2+ level. Serum magnesium is known to be act as a cofactor for the activation of Na+ K+ ATPase [42,43]. Hence, in this observation, decreased serum Mg2+ level could also be the reason for inhibition of Na+ K+ ATPase activity. The decrease in the levels of ATPases by the free radicals in the aspartame administered animals could be due to free radical induced cell damage by methanol metabolite of aspartame and their severe cytotoxic effects, such as lipid peroxidation in cell membrane followed by the alteration of the membrane fluidity, enzyme properties and ion transport. Enhanced lipid peroxidation which may act on the sulphhydryl groups present in the active sites of the ATPases [44]. Since the membrane bound enzymes are ‘SH’ group containing enzymes [45] and these enzymes are extremely sensitive to hydroperoxides and superoxide radicals [46]. Thiol modification (i.e. loss of protein sulfhydryl group) has been recognized as a critical event in cytotoxicity [47]. The present study clearly confirms that aspartame can be act as chemical stressor as indicated by the elevated corticosteroid level in the aspartame treated group. Increased corticosterone has been shown to decrease the size and weight of the spleen and thymus. The significant reduction in organ weight and organ cell count may
be due to oxidative damage which is studied by Skrzydlewksa and Szynaka [48] who reported that oxidative damage caused marked organ weight loss in albino rats upon methanol intoxication (this is also reported by Parthasarathy et al., [34]). Formaldehyde, the first metabolite of methanol increases the population of shrunken cells, dead cells and hydolipid cells [49] which might be the reason for decreased cellularity (reduction in organ weight and cell count) as with our observation. 5. Conclusion The results of present study clearly point out that aspartame induce oxidative stress by generation of free radicals. Such induced oxidative stress result in inhibiting function of ion-dependent Atpase’s leads to disruption in ion homeostasis, resulting altered cellular metabolism, change in cell membrane fluidity and disturbance of vital function of immune organs which also decreased the cellularity (reduction in organ weight and cell count) of these organs. Aspartame metabolite methanol or formaldehyde may be the causative factors behind the changes observed. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgments The authors gratefully acknowledge the University of Madras for their financial support (UGC No.D.1. (C)/TE/2012/1868). The authors acknowledge Mr. Sunderaswaran loganathan & Mr. Annadurai for their constant support and help. References [1] Food Drug Administration (FDA). Aspartame: Commissioner’s final decision. Fed Reg 1981;46:38285–308. [2] Food Drug Administration (FDA). Food additives permitted for direct addition to food for human consumption: Aspartame. Fed Reg 1983;48:31376–82.
Please cite this article in press as: Choudhary AK, Rathinasamy SD. Effect of long intake of aspartame on ionic imbalance in immune organs of immunized wistar albino rats. Biomed Aging Pathol (2014), http://dx.doi.org/10.1016/j.biomag.2014.03.001
G Model BIOMAG-137; No. of Pages 7
ARTICLE IN PRESS A.K. Choudhary, S.D. Rathinasamy / Biomedicine & Aging Pathology xxx (2014) xxx–xxx
[3] Food Drug Administration (FDA). Food additives permitted for direct addition to food for human consumption: aspartame. Fed Reg 1996;61: 33654–6. [4] European Food Safety Authority (EFSA). Opinion of the Scientific Panel on food additives, flavourings, processing aids and materials in contact with food (AFC) on a request from the Commission related to a new long-term carcinogenicity study on aspartame. Question number EFSA-Q-2005-122. Adopted on 3 May 2006. EFSA J 2006;356:1–44. [5] Trocho C, Pardo R, Rafecas I, Virgili J, Remesar X, Fernandez-Lopez JA, et al. Formaldehyde derived from dietary aspartame binds to tissue components in vivo. Life Sci 1998;63:337–49. [6] Mattews DM. Absorption of peptides, amino acids, and their methylated derivatives. In: Stegink LD, Filer Jr LJ, editors. Aspartame physiology and biochemistry. New York: Dekker; 1984. p. 29–46. [7] Haschemeyer RH, Haschemeyer AEV. Proteins. New York: John Wiley & Sons; 1973. p. 11–30. [8] Metzler DE. Biochemistry. New York: Academic Press; 1977. p. 90–107. [9] Kosawer NJ, Zipser Y, Faltin Z. Membrane thiol disulfide status in glucose6-phosphate dehydrogenase deficient red cells. Relationship to cellular glutathione. Biochim Biophys Acta 1991;1058:152–60. [10] Stekhoren MA. Bonting SL. Transport ATPases: properties and functions. Physiol Rev 1981;61:1–76. [11] Lopaczyski W, Zeisel SH. Antioxidants, programmed cell death, and cancer. Nutr Res 2001;21:295–307. [12] McIntosh LJ, Sapolsky RM. Glucocorticoids increase the accumulation of reactive oxygen species and enhance adriamycin-induced toxicity in neuronal culture. Exp Neurol 1996;141:201–6. [13] McEwen BS. The neurobiology of stress: from serendipity to clinical relevance (1). Brain Res 2000;886:172–89. [14] Mastorakos G, Ilias I. Maternal hypothalamic-pituitary-adrenal axis in pregnancy and the postpartum period, Postpartumrelated disorders. Ann N Y Acad Sci 2000;900:95–106. [15] Mourad M. Effect of aspartame on some oxidative stress parameters in liver and kidney of rats. AJPP 2011;5(6):672–8. [16] Maker AB, Tephly TR. Methanol in poisoning folate deficient rats. Nature 1976;261:715–6. [17] Tephly TR. The toxicity of methanol. Life Sciences 1991;48:1031–41. [18] Ming H, Wang SY, Craig A, Meadows Shirley W. Thenen methotrexate effects on folate status and the deoxyuridine suppression test in rats. Nut Re 1989;9:431–44. [19] Rabinowitz JC, Pricer WE. Formiminotetrahydrofolic acid and methenyltetrahydrofolic acid as intermediates in the formation of N10-formyltetrahydrofolic acid. J Am Chem Soc 1956;78(21):5702–4. [20] Feldman S, Conforti N. Participation of dorsal hippocampus in the glucocorticoids feedback effect on adrenocortical activity. Neuroendocrinology 1980;30:52–5. [21] Clark I. A colometric reaction for the estimation of cortisone, hrdrocortisone, aldosterone and related steroids. Nature 1955;75:123–4. [22] Bonting SL. Sodium potassium activated adenosine triphosphatase and cation transport. In: Bitler EE, editor. Membrane and ion transport, 1. London: Interscience Wiley; 1970. p. 257–63. [23] Hjerten S, Pan H. Purification and characterization of two forms of a low affinity Ca2+ ATPase from erythrocyte membranes. Biochim Biophys Acta 1983;728:281–8. [24] Ohnishi T, Suzuki T, Suzuki Y, Ozawa K. A comparative study of plasma membrane Mg2+ ATPase activities in normal, regenerating and malignant cells. Biochim Biophys Acta 1982;684:67–74. [25] Fiske CH, Subbarow Y. The colorimetric determination of phosphorous. J Biol Chem 1925;663:75–400.
7
[26] Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265–75. [27] Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 1979;95:351–8. [28] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248–54. [29] Cross RJ, Brooks WH, Roszman TL. Hypothalamic immune interaction. J NeurolSci 1982;53:557–66. [30] Pragasam V, Kalaiselvi P, Sumitra K, et al. Counterction of oxalate induced nitrosative stress by supplementation of L- arginine, a potent antilithic agent. Clin Chim Acta 2005;354:159–66. [31] Gubdjorson S, Hallgrimson J, Skuldottir G. Arterial pollution. In: Peter H, Geshaw GA, Paoethi, editors. Properties of transport adenosine triphosphatase. New York: Plenum Publishing Corp; 1983. p. 101. [32] Stefano GB, Fricchione GL, Slingsby BT, Benson H. The placebo effect and relaxation response: neural processes and their coupling to constitutive nitric oxide. Brain Res Brain Res Rev 2001;35(1):1–19. [33] Weglicki WB, Dickens BF, Mak IT. Enhanced lysosomal phospholipid degradation and lysphospholipid production due to free radical. Biochem Biophys 1984;124:229–35. [34] Parthasarathy JN, Ramasundaram SK, Sundaramahalingam M, et al. Methanol induced oxidative stress in ratlymphoid organs. J Occup Health 2006;48:20–7. [35] Zararsiz I, Sarsilmaz M, Tas U, et al. Protective effect of melatonin against formaldehyde-induced kidney damage in rats. Toxicol Ind Health 2007;23(10):573–9. [36] Jaeschke H. The role of reactive oxygen species in hepatic ischaemiareperfusion injury and preconditioning. J Inv Surg 2003;16:127–40. [37] Contreras RG, Shoshani L, Flores-Maldonado CA, et al. Relationship between Na+K+ Atpase and cell attachment. J Cell Sci 1999;112:4223–32. [38] Matels JM, Gomes CP, De Castro IN. Antioxidant enzymes and human disease. Biochem Pharmacol 1999;28:251–3. [39] Berridge MJ, Lipp P, Bootman MD. The versatility and universality of calcium signaling. Nat Rev Mol Cell Biol 2000;1:11–21. [40] Jain KS, Shohet SB. Calcium potentiates the peroxidation of erythrocytemembrane lipids. Biochem Biophys Acta 1981;642:46–54. [41] Sanu H, Rubin H. Ions, cell proliferation and cancer. In: The role of magenisum in cell proliferation and transformation. New York: Academic press; 1982. p. 517–37. [42] Ebel H, Gunther T. Role of magnesium in cardiac diseases. J Clin Biochem 1983;21:249. [43] Altura BM, Altura BT. New perspectives on the role of magnesium in the pathophysiology of cardiovascular system. Clinical aspects. Magnesium 1985;4:226. [44] Pragasam S, Jenitha V, Kalaiselvi X, et al. Oxidative stress a predisposing factor for renal stone formation – amelioration by vitamin E supplementation. Clin Chem Acta 2004;350:57–63. [45] Upasani CD, Balaraman R. Effect of vitamin E, vitamin C and spirulina on the levels of membrane bound enzymes and lipids in some organs of rats exposed to lead. Ind J Pharmacol 2001;33:185–91. [46] Jain KS, Shohet SB. Calcium potentiates the peroxidation of erythrocyte membrane lipids. Biochem Biophys Acta 1981;642:46–54. [47] Pasoe GA, Olafsdottir K, Reed DJ. Vitamin E protection against chemical induced injury. I. Maintenance of cellular protein thiols as a cytoprotective mechanism. Arch Biochem Biophy 1987;256:150–68. [48] Skrzydlewksa E, Szynaka B. Ultrastructral elevation of lysosome and biochemical change in cathepsin D distribution in hepatocytes in methanol intoxication. Roczniki Akademii Medycznej W Bialymstoku 1997;42:47–55. [49] Nakao H, Umebayashi C, Nakata M, Nishizaki Y, Noda K, Okanao Y, et al. Formal dehyde induced shrinkage of rat thymocyte. J Pharmacol Sci 2003;91:83–6.
Please cite this article in press as: Choudhary AK, Rathinasamy SD. Effect of long intake of aspartame on ionic imbalance in immune organs of immunized wistar albino rats. Biomed Aging Pathol (2014), http://dx.doi.org/10.1016/j.biomag.2014.03.001