Mechanistic insights in aspartame-induced immune dysregulation

Arbind Kumar Choudhary*1, Yeong Yeh Lee2

1Department. of Physiology, Government Medical College, Shivpuri, (M.P.) India.

2School of Medical Sciences, Universiti Sains Malaysia, Kota Bharu, Kelantan, Malaysia

*Corresponding author Dr. Arbind Kumar ChoudharyDept. of PhysiologyGovernment Medical College, Shivpuri (M.P.), India. Ph: +918435969782/+918839748106 Email: arbindchoudhary111@gmail.com

1

Abstract

Background & objective: Aspartame, (L-aspartyl-L-phenylalanine methyl ester) is a widely

used artificial sweetener but studies raise safety concerns of aspartame metabolites

especially methanol. In this review, we aimed to provide mechanistic insights that may explain

aspartame-induced immune dysregulation.

Findings: While evidence is limited, from available literatures, possible mechanisms for immune

dysfunction associated with aspartame include (1) alterations in bidirectional communication

between neuro-immune-endocrine responses (2) disruption of the brain-gut-microbiota-immune

axis (3) induction of oxidative stress in immune cells and organs and lastly (4) the immune-

activation effect of methanol.

Conclusion: Further studies are needed to confirm above proposed mechanisms that may

explain aspartame-induce immune dysregulation.

Keyword: Aspartame; immune regulation; oxidative stress; guts dysbiosis.

2

Introduction

Non-nutritive artificial sweeteners including aspartame, saccharin, sucralose, acesulfame-K and

neotame are popular alternatives to sucrose for weight control [1-3]. Aspartame is available

commercially under different market labels such as Nutra sweetTM, CanderelTM, and EqualTM [4].

Aspartame is consumed by millions of people around the world and is used in soft drinks,

dessert mixes, frozen desserts and yogurt, chewable multi-vitamins, breakfast cereals and

medicines [5]. Despite initial popularity, it has now become a possible global public health

concern[6, 7] because of its induction of neurophysiological symptoms[8] and immunological

side-effects[9-13]. There is increasing evidence that aspartame may induce metabolic

consequences [14].

After ingestion, aspartame is immediately absorbed from small intestinal lumen and metabolized

by gut esterases and peptidases before entering circulation. Aspartame is hydrolyzed into its

three basic components: two amino acids i.e. aspartic acid and phenylalanine and methanol

[15]. On a weight basis, metabolism of aspartame generates approximately 50% phenylalanine,

40% aspartic acid and 10% methanol [16]. Aspartic acid is then transformed into alanine and

oxaloacetate; phenylalanine mainly into tyrosine and lesser extent into phenylethylamine and

phenylpyruvate [17] and methanol into formaldehyde and then formic acid [18].

Despite being around for long, the long-term safety of aspartame is still debated due to

inconsistent toxicological and clinical studies [19]. Normal and excessive use of aspartame may

have adverse effects within the human body due to buildup of its metabolites in tissues including

the brain [20]. Rising incidence of brain tumors has been linked to aspartame consumption in

epidemiology studies [21, 22] but these have to be interpreted with caution due to their

methodology limitations [19]. Early studies have associated aspartame with allergic reactions

[23] but these findings were later contradicted [24]. More recent studies using animals models

have revealed that aspartame activated the hypothalamic– pituitary–adrenal (HPA) axis [10, 25],

the sympathetic nervous system (SNS) [26, 27] and induced oxidative stress in immune organs

[10-13].

The immune system communicates bi-directionally with the brain and endocrine system [28].

Various chemicals or immunomodulators target the immune system and a chemical insult to the

immune system can result in enhancement or suppression of immune response [29]. Immune

regulation is a composite balance between regulatory and effector cells [30]. Interfering with

immunomodulation process may result in either immunostimulation or immunosuppression [31].

3

In this review, we discuss the interactions between aspartame and the immune system and

provide mechanistic insights that may explain the immune dysregulation associated with

aspartame and its health consequences.

4

Interactions between aspartame and the neural-immune-endocrine system

Chronic aspartame consumption has been linked to neuro-degeneration and apoptosis in mice

brains [32-36]. Although exact mechanisms are not entirely clear but impaired regulation

between aspartame and the neural-immune-endocrine system may be responsible. Most

probably, aspartame acts as a chemical stressor that activates (1) the HPA axis [10, 25] with

subsequent increases in stress hormone (cortisol) levels and (2) the sympathetic–adrenal–

medullary (SAM) axis [26, 27] that disrupts the sympathetovagal balance (Figure:1A&B).

Stress hormones from activation of HPA axis then (1) bind and activate glucocorticoid receptors

within cytoplasm of immune T and B cells [37-39] which (2) through genomic or non-genomic

activation, cause the release of cytokines (including interferon-γ (IFN-γ), interleukin-1 (IL-1), IL-

2, IL-6 and tumor-necrosis factor (TNF)) (Figure 1c) [28] and (3) imbalance of anti-inflammatory

cytokines (IL-1 receptor antagonist, IL-6) and proinflammatory cytokines (specific cytokine

receptors for IL-1, TNF – α) lead to immune-mediated inflammatory responses. In vitro, levels of

inflammatory mediator IL-6, vascular endothelial growth factor (VEGF) and their soluble

receptors from endothelial cells have been shown to increase due to aspartame [37]. The

resultant immune response may be a direct effect of aspartame or its metabolite methanol or

formaldehyde but till date, the exact molecule (s) remains uncertain.

Of the immune cells involved in aspartame-mediated responses, T helper 1 (TH1) and T helper 2

(TH2) cells are most important. TH1cells are involved in pro-inflammatory responses and TH2

cells in anti-inflammatory responses, and they function in a homeostatic relationship i.e. when

the TH1 cells become overactive, they can suppress activity of TH2 cells and vice versa. The

above explains the observation in rats that oral administration of aspartame for 90-days first

induces an immune shift from production of TH1 to TH2 cells, probably to suppress TH1 pro-

inflammatory responses [11].

In addition, cytokines release associated with aspartame stress may alter local glucocorticoid

availability and glucocorticoid receptor (GR) function [38] and consequently cortisol

receptiveness. Cortisol receptiveness has an important influence on susceptibility and

resistance to inflammation. Low levels of cortisol may lead to an impaired glucocorticoid

response that predisposes to autoimmune and inflammatory diseases [39]. At present, the

amount of aspartame that interferes with cortisol receptiveness is not well-defined.

5

6

A

Figure 1 A & B: Aspartame and the neuro-immuno-endocrine regulation: Aspartame and associated metabolites may interrupt the neuro-immuno-endocrine regulation by interfering cytokines and neurotransmitters release through activation of the hypothalamic–pituitary–adrenal (HPA) axis and the sympathetic–adrenal–medullary (SAM) axis.

7

B

Figure 1C: Proposed mechanism which may link aspartame with post-translational modification of the glucocorticoid receptor: Aspartame consumption leads to elevated cortisol which binds to the glucocorticoid receptor, undergoes genomic and non-genomic

8

C

activation and may lead to induction/suppression of anti-inflammatory (IL-1 receptor antagonist, IL-6) / inflammatory (IL-1, TNF – α) proteins.

9

Interactions between aspartame and the brain-gut-microbiota-immune axis

In a bidirectional fashion, the brain communicates with the gastrointestinal (GI) tract through the

autonomic nervous system, the HPA axis and the gut microbiota to modulate mucosal immune

functions [40, 41] . In a nutshell, gut is like ‘tentacles’ for the brain to ‘sample’ the environment

similar with the eyes being the ‘window’ for the brain to ‘see’ the environment. Aspartame may

disrupt the brain-gut-microbiota axis resulting in immune dysregulation through several

mechanisms (Figure 2A & 2B).

Neurophysiological symptoms due to aspartame [8] seem to implicate intestinal dysbiosis [42].

Exact mechanisms for dysbiosis are not clear but metabolites of aspartame especially methanol

may be implicated. Catecholamines and cortisol associated with aspartame stress on the

nervous system [43] can result in changes of microbiota composition but also impaired gut

barrier function [42]. Increased gut permeability allows bacteria and bacterial antigens to cross

the epithelial barrier and activate pro-inflammatory mucosal immune responses.

Aspartame itself has been shown to increase abundance of Enterobacteriaceae, known to

cause inflammation and insulin resistance [44] although this was not replicated by Frankenfeld

et al [45]. The aspartame-induced dysbiosis affect the brain through synthesis of microbial by-

products that can gain access to the brain via bloodstream but also through cytokine release

from mucosal immune cells, release of gut hormones such as 5-hydroxytryptamine (5-HT) from

entero-endocrine cells, or via afferent neural pathways, including the enteric nervous system

[46-49].

10

11

A

Figure 2A&B: Aspartame and modulation of the bi-directional gut-brain-immune axes: Aspartame use leads to oxidative stress in the brain, causing neurophysiological symptoms and intestinal dysbiosis. Immune regulation in aspartame consumers is affected through dysbiosis which may disrupts the synthesis of microbial by-products and precursors (fatty acid, GABA, 5-HT precursor) that gain access to the brain via the bloodstream. For example, cytokines are released from mucosal immune cells (dendritic cell) and gut hormones, 5-hydroxytryptamine (5-HT) are released from entero-endocrine cells and are transported via afferent neural pathways, including the enteric nervous system.

12

B

Interactions between aspartame and oxidative stress and immune regulation

Oxidative stress is indicated by an imbalance between pro-oxidant production and antioxidant

defenses [50]. Immune cells use reactive oxygen species (ROS) to support their functions and

need acceptable levels of antioxidant defenses to avoid the destructive effect of excessive ROS

production [51]. The immune system has various regulatory functions that depend on oxidant-

antioxidant balance. Oxidative stress from aspartame causes excess ROS in immune cells or

organs (thymus, bone marrow, spleen, lymph nodes) and ultimately immune dysfunction (either

immune-activation or immune-suppression) (Figure 3) [52, 53]. The production of excess ROS

is possibly associated with production of methanol [10, 36, 54]. Aspartame alone or in the

presence of mild systemic inflammatory response increases oxidative stress and inflammation

in the brain but not in the liver [55].

Immune activation due to aspartame is associated with increased in pro-inflammatory cytokines,

acute phase protein, adhesion molecules and chemokines (Figure 3). Excess ROS affects

integral membrane function including cell-mediated immune responses such as antigen

reception, secretion of lymphokines and antibodies, contact cell lysis, and lymphocyte

transformation [56]. Leukocytes adhere to endothelial cells when excess ROS is present [57].

Significant neutrophil infiltration was found in liver parenchyma with methanol [60, 61] and this is

probably mediated through enhanced chemokine release from Kupffer cells or hepatic

sinusoidal cells and maybe glutathione-dependent [62]. Adhesion of leukocytes to endothelial

cells is an early step in inflammation and depends on expression of cell-surface receptors

known as cell adhesion molecules [58]. Excess ROS also cause direct tissue injury to cell

membranes, lipids, proteins and DNA [59]. IL-6 is produced at the site of inflammation and plays

a key role in the production of most acute phase protein [63]. In vitro, levels of pro-inflammatory

IL-6 have been shown to increase after aspartame administration [37].

Immunosuppression due to aspartame is associated with reduced leucocyte migration,

phagocytosis, intracellular killing and chemotaxis (Figure 3). The adhesion molecule β2

integrins stored in cell granules of neutrophils are up- regulated for a firm adhesion during

margination process [65]. In the presence of methanol, there was decrease in adherence of

neutrophils which may be due to either internalization or increased shedding of β2 integrins [53,

66]. In addition, oxidative stress decreases release of lymphokines from antigen-sensitized T-

lymphocytes and this could result in decreased mobilization of immune effector cells [66].

Oxidative stress has been shown to decrease mobilization of leukocytes to the site of immune

13

challenge leading to the suppression of delayed type hypersensitivity reactions (cutaneous foot

pad thickness (FPT) reaction) [66].

Figure 3: Aspartame usage may cause oxidative damage and immune dysfunction (immunoactivation /immunosuppression)

However, the role of aspartame as a cause of immune dysfunction is not straightforward.

Elevated heat shock protein (Hsp70) expression has been shown to prevent apoptosis and

enhanced the survival of immune organs after 90-days of oral aspartame consumption [67].

However, the effect of aspartame on apoptosis process in cancer is poorly understood. Recently

in one study, HeLa cells were exposed to different concentrations (0.01–0.05 mg/ml) of

aspartame for 48 h, where it slowed down the apoptosis process [68]. While in other study on

the mice hippocampus, aspartame has neurotropic effect [69].

14

Aspartame may have a therapeutic value in 2, 4-dinitrofluorobenzene (DNFB) evoked atopic

dermatitis–like symptoms shown in NC/Nga mice through inhibition of infiltration of inflammatory

cells (eosinophils, mast cells, and CD4+ T cells), and suppressed the expression of cytokines

(IL-4 and IFN-γ, and total serum IgE levels) [66]. Another example is that aspartame can inhibit

the binding of IgG in a substantial proportion of IgM rheumatoid factors (RFs). Interference of

RF reactivity, especially in rheumatoid arthritis patients may alleviate the pain and immobility

resulting from chronic inflammation of the joints [70].

Interactions between aspartame by-product, methanol and immune regulation

Methanol exposure in aspartame consumers may occur when (1) soft drinks are stored for

longer periods or at high temperatures and the aspartame begins to decompose and (2) when

aspartame is metabolized in the small intestine and the methyl group of the dipeptide is

hydrolysed by chymotrypsin [71]. The toxicity of methanol and its metabolites (formaldehyde

and formate) after aspartame ingestion have been observed at both normal and abuse

dosages (Table 1).

In humans, only abuse doses of aspartame (above 100 mg/kg) can elevate blood methanol

concentrations to significant levels, but at normal doses the methanol can still remain detectable

for eight or more hours after ingestion [72, 73]. Daily intake of methanol from natural sources is

less than 10 mg. Aspartame sweetened beverages contain 55 mg of methanol per liter, and it is

nearly double than in some carbonated sodas. Aspartame is present in many products

worldwide and is not limited by calories or osmolality and intake can equal the daily water loss

of an individual. Thus, consuming unlimited aspartame sweetened soft drinks, unintentionally

increasing their intake [74]. Under such circumstances, daily methanol intake may rise to toxic

levels [71]

Oral administration of aspartame causes elevated blood methanol levels in rats [25], which is

rapidly absorbed and metabolized (oxidized) into formaldehyde or formic acid which later

accumulate in various tissues [75]. Rats metabolize methanol faster than humans and formate

does not accumulate in rat tissues [76]. Hepatic tetrahydrofolate levels in humans and monkeys

are only half of those in rats [77]. Human being has very low hepatic folate content [78]. Hence

in folic acid deficiency, methanol metabolism could take the alternate pathway (microsomal

pathway) [79]. Methanol is more toxic to humans than rats or other small laboratory animals

15

because humans has limited key enzymes such as tetrahydrofolate that are required for

methanol metabolism [80, 81].

Aspartame may cause allergic reactions, including anaphylaxis, due to its metabolites [82]. The

formation of formaldehyde adduct, which binds with proteins and accumulates in tissues rather

than metabolizing into formate, may be particularly hazardous [75] Formate allergies affect

certain tissues, including the retina. Visual deterioration (including transient blindness diagnosed

as optic neuritis) has been observed because of excessive intake of aspartame [83, 84].

Although exact mechanism is unknown but it has been postulated that formaldehyde from

aspartame breakdown could possibly trigger a systemic form of chronic eyelid dermatitis in

formaldehyde-sensitive patients [85, 86], however this theory is later disputed [87].

Formate/formaldehyde could compromise immune regulation through immune activation,

autoantibodies, and higher antibody titers to anti-HCHO-HAS antibodies [isotypes of anti-

formaldehyde (HCHO) conjugated to human serum albumin (HAS)] [88].

16

Table 1: Studies on the aspartame metabolite, methanolS.no Study design Result Conclusion References

Human studies1. Aspartame administered at daily

ingestion dose (34 mg/kg body weight) and abuse doses (100, 150, and 200 mg/kg body weight) in normal adult subjects

The blood methanol concentrations were below the level of detection at 34 mg/kg body weight and were significantly elevated after ingestion of each abuse dose.

No significant increase in blood formate concentrations over predosing concentrations was noted.

[73]

2. Blood methanol concentrations were measured in 1-year-old infants administered aspartame dose for adults (34, 50 and 100 mg/kg body weight)

Methanol concentrations were below the level of detection in the blood administered at 34 mg/kg body weight, but were significantly elevated at 50 and 100 mg/kg body weight.

No significant increase in blood formate concentrations at 34 mg/kg body weight

[89]

3. Four male adult volunteers each received 500 mg aspartame, equivalent to 6–8.7 mg/kg bodyweight. This was approximately the FDA’s estimate of mean daily human consumption.

A significant rise in serum methanol level was observed at 45min, after administration and returned to basal values at 2 hr after treatment.

The temporary serum methanol increases showed that peak values are within the range of FDA’s estimated doses.

[90]

17

4. Six normal young adults ingested eight successive servings of unsweetened and aspartame sweetened beverage (600 mg); a dose equivalent to 36 oz of aspartame sweetened diet beverage.

Blood methanol and formate concentrations remained within normal limits.

Aspartame is metabolized completely when administered at levels that may be ingested by normal individuals who are heavy users of diet beverages.

[91]

Animal studies5. The dose given to rats was the FDA’s

approval human dose (34 mg/kg. bodyweight).

A significant rise in serum methanol level was observed at 1 hr, after administration and returned to basal values at 4 hr after treatment.

There are temporary serum methanol increases but peak values are within the range of individual basal levels.

[90]

6. Aspartame toxicity, the effects of methanol and its metabolites (formaldehyde and formate) on dissociated rat thymocytes.

Threshold concentrations of formaldehyde (a metabolite of methanol), were slightly higher than the blood concentrations of methanol previously reported in subjects administered abuse doses of aspartame.

Aspartame at abuse doses is harmless to humans.

[92]

7. The oral administration of aspartame (75 mg/kg b.wt) for 90-days in rats

The plasma methanol level was significantly increased in aspartame treated rats.

The chronic exposure of aspartame results in detectable methanol levels in blood.

[25]

8. The FDA approved aspartame dosage 40 mg/kg b.wt were given daily for 90 days in rats .

Aspartame treated animals showed a significant increase in plasma methanol/ formate level.

Increased methanol/ formate level may be an indication of oxidative damage caused by aspartame metabolites.

[34, 35]

18

19

Conclusion

Aspartame consumption can disrupt the immune responses by causing dissociation between (1) bidirectional communication between neuro-immune-endocrine responses (2) bidirectional brain-gut-microbiota-immune axis (3) induction of oxidative stress in immune cells and organs and (4) elevated levels of aspartame metabolite, methanol.

We conclude that, at this stage, the exact mechanisms for aspartame-induced immune dysfunction are not entirely clear but in this review we have proposed a number of potential mechanisms. However, future studies are needed to confirm that aspartame and its metabolites are the real culprits in causing immune dysregulation.

Funding: The authors greatly acknowledge Research University Individual (RUI) Grant from

Universiti Sains Malaysia (1001/PPSP/812151).

Disclosure

The authors do not have any conflicts of interest to declare.

References

1. Anton, S.D., et al., Effects of stevia, aspartame, and sucrose on food intake, satiety, and postprandial glucose and insulin levels. Appetite, 2010. 55(1): p. 37-43.

2. Mattes, R.D. and B.M. Popkin, Nonnutritive sweetener consumption in humans: effects on appetite and food intake and their putative mechanisms. The American journal of clinical nutrition, 2009. 89(1): p. 1-14.

3. Oku, T. and S. Nakamura, Digestion, absorption, fermentation, and metabolism of functional sugar substitutes and their available energy. Pure and Applied Chemistry, 2002. 74(7): p. 1253-1261.

4. Grenby, T., Intense sweeteners for the food industry: an overview. Trends in Food Science and Technology, 1991. 2: p. 2-6.

5. Butchko, H.H. and W.W. Stargel, Aspartame: scientific evaluation in the postmarketing period. Regulatory Toxicology and Pharmacology, 2001. 34(3): p. 221-233.

6. Choudhary, A., Aspartame: should individuals with Type II Diabetes be taking it? Current diabetes reviews, 2017.

7. Choudhary, A.K. and E. Pretorius, Revisiting the safety of aspartame. Nutrition reviews, 2017. 75(9): p. 718-730.

8. Choudhary, A.K. and Y.Y. Lee, Neurophysiological symptoms and aspartame: What is the connection? Nutritional neuroscience, 2017: p. 1-11.

9. Abbott, N.J., L. Rönnbäck, and E. Hansson, Astrocyte–endothelial interactions at the blood–brain barrier. Nature Reviews Neuroscience, 2006. 7(1): p. 41-53.

20

10. Choudhary, A.K. and R.S. Devi, Imbalance of the oxidant-antioxidant status by aspartame in the organs of immune system of Wistar albino rats. African Journal of Pharmacy and Pharmacology, 2014. 8: p. 220-230.

11. Choudhary, A.K. and R.S. Devi, Longer period of oral administration of aspartame on cytokine response in Wistar albino rats. Endocrinología y Nutrición, 2015. 62(3): p. 114-122.

12. Choudhary, A.K. and S.D. Rathinasamy, Aspartame induces alteration in electrolytes homeostasis of immune organs in wistar albino rats. Biomedicine and Preventive Nutrition, 2014. 4(2): p. 181-187.

13. Choudhary, A.K. and S.D. Rathinasamy, Effect of long intake of aspartame on ionic imbalance in immune organs of immunized wistar albino rats. Biomedicine and Aging Pathology, 2014. 4(3): p. 243-249.

14. Choudhary, A. and E. Pretorius, Revisiting the Safety of Aspartame. . Nutritional Review (Accepted). , Forthcoming 2017.

15. Stegink, L.D. and L. Filer, Effects of aspartame ingestion on plasma aspartate, phenylalanine and methanol concentrations in normal adults. The clinical evaluation of a food additive. Assessment of Aspartame. Tschanz C, Butchko HH, Stargel WW, Kotsonis FN, Edts, 1996: p. 67-86.

16. Humphries, P., E. Pretorius, and H. Naude, Direct and indirect cellular effects of aspartame on the brain. European Journal of Clinical Nutrition, 2008. 62(4): p. 451-462.

17. Stegink, L.D., et al., Repeated ingestion of aspartame-sweetened beverage: effect on plasma amino acid concentrations in individuals heterozygous for phenylketonuria. Metabolism, 1989. 38(1): p. 78-84.

18. Opperman, J., Aspartame metabolism in animals. Aspartame Physiology and Biochemistry (Stegink LD, Filer LJ Jr, eds). New York: Dekker, 1984: p. 141-159.

19. Magnuson, B., et al., Aspartame: a safety evaluation based on current use levels, regulations, and toxicological and epidemiological studies. Critical reviews in toxicology, 2007. 37(8): p. 629-727.

20. Renwick, A., The fate of intense sweeteners in the body. Food chemistry, 1985. 16(3-4): p. 281-301.

21. Olney, J.W., et al., Increasing brain tumor rates: is there a link to aspartame? Journal of Neuropathology & Experimental Neurology, 1996. 55(11): p. 1115-1123.

22. Weihrauch, M.R. and V. Diehl, Artificial sweeteners—do they bear a carcinogenic risk? Annals of Oncology, 2004. 15(10): p. 1460-1465.

23. Atkins, F.M. and D.D. Metcalfe, The diagnosis and treatment of food allergy. Annual review of nutrition, 1984. 4(1): p. 233-255.

24. Szucs, E., K. Barrett, and D. Metcalfe, The effects of aspartame on mast cells and basophils. Food and chemical toxicology, 1986. 24(2): p. 171-174.

25. Iyyaswamy, A. and S. Rathinasamy, Effect of chronic exposure to aspartame on oxidative stress in brain discrete regions of albino rats. Journal of Biosciences, 2012. 37(4): p. 679-688.

26. Choudhary, A.K., L. Sundareswaran, and R.S. Devi, Aspartame induced cardiac oxidative stress in Wistar albino rats. Nutrition Clinique et Métabolisme, 2016. 30(1): p. 29-37.

27. Choudhary, A.K., L. Sundareswaran, and R.S. Devi, Effects of aspartame on the evaluation of electrophysiological responses in Wistar albino rats. Journal of Taibah University for Science, 2016. 10: p. 505-512.

28. Glaser, R. and J.K. Kiecolt-Glaser, Stress-induced immune dysfunction: implications for health. Nature Reviews Immunology, 2005. 5(3): p. 243-251.

29. Colosio, C., et al., Low level exposure to chemicals and immune system. Toxicology and applied pharmacology, 2005. 207(2): p. 320-328.

21

30. Bluestone, J.A. and A.K. Abbas, Natural versus adaptive regulatory T cells. Nature Reviews Immunology, 2003. 3(3): p. 253-257.

31. Mazmanian, S.K., et al., An immunomodulatory molecule of symbiotic bacteria directs maturation of the host immune system. Cell, 2005. 122(1): p. 107-118.

32. Abdel-Salam, O.M., N.A. Salem, and J.S. Hussein, Effect of aspartame on oxidative stress and monoamine neurotransmitter levels in lipopolysaccharide-treated mice. Neurotoxicity Research., 2012. 21(3): p. 245-255.

33. Abhilash, M., et al., Long-term consumption of aspartame and brain antioxidant defense status. Drug and Chemical Toxicology, 2013. 36(2): p. 135-140.

34. Ashok, I. and R. Sheeladevi, Biochemical responses and mitochondrial mediated activation of apoptosis on long-term effect of aspartame in rat brain. Redox Biology, 2014. 2: p. 820-831.

35. Ashok, I. and R. Sheeladevi, Neurobehavioral changes and activation of neurodegenerative apoptosis on long-term consumption of aspartame in the rat brain. Journal of Nutrition and Intermediary Metabolism, 2015. 2(3): p. 76-85.

36. Ashok, I., R. Sheeladevi, and D. Wankhar, Long term effect of aspartame (artificial sweetener) on membrane homeostatic imbalance and histopathology in the rat brain. Free Radicals and Antioxidants, 2013. 3: p. S42-S49.

37. Alleva, R., et al., In vitro effect of aspartame in angiogenesis induction. Toxicology in vitro, 2011. 25(1): p. 286-293.

38. Kaestner, F., et al., Different activation patterns of proinflammatory cytokines in melancholic and non-melancholic major depression are associated with HPA axis activity. Journal of affective disorders, 2005. 87(2): p. 305-311.

39. Sternberg, E.M., Neural regulation of innate immunity: a coordinated nonspecific host response to pathogens. Nature Reviews Immunology, 2006. 6(4): p. 318-328.

40. Collins, S.M. and P. Bercik, The relationship between intestinal microbiota and the central nervous system in normal gastrointestinal function and disease. Gastroenterology, 2009. 136(6): p. 2003-2014.

41. Romijn, J.A., et al., Gut–brain axis. Current Opinion in Clinical Nutrition & Metabolic Care, 2008. 11(4): p. 518-521.

42. Choudhary, A.K. and Y. Lee, Dysregulated microbiota-gut-brain axis: Does it explain aspartame metabolic and other side-effects? Nutrition & Food Science, 2017. 47(5): p. 648-658.

43. Groot, J., et al., Stress Induced Decrease of the Intestinal Barrier Function: The Role of Muscarinic‐ Receptor Activation. Annals of the New York Academy of Sciences, 2000. 915(1): p. 237-246.

44. Palmnäs, M.S., et al., Low-dose aspartame consumption differentially affects gut microbiota-host metabolic interactions in the diet-induced obese rat. PLoS One, 2014. 9(10): p. e109841.

45. Frankenfeld, C.L., et al., High-intensity sweetener consumption and gut microbiome content and predicted gene function in a cross-sectional study of adults in the United States. Annals of epidemiology, 2015. 25(10): p. 736-742. e4.

46. Bonaz, B. and J.-M. Sabaté, [Brain-gut axis dysfunction]. Gastroentérologie clinique et biologique, 2009. 33: p. S48-58.

47. Hooper, L.V., D.R. Littman, and A.J. Macpherson, Interactions between the microbiota and the immune system. Science, 2012. 336(6086): p. 1268-1273.

48. Mulak, A. and B. Bonaz, Irritable bowel syndrome: a model of the brain-gut interactions. Medical science monitor, 2004. 10(4): p. RA55-RA62.

49. Thakur, A.K., et al., Gut-microbiota and mental health: current and future perspectives. J Pharmacol Clin Toxicol, 2014. 2: p. 1016-1031.

50. Finkel, T. and N.J. Holbrook, Oxidants, oxidative stress and the biology of ageing. Nature, 2000. 408(6809): p. 239-247.

22

51. Victor, V.M., M. Rocha, and M. De la Fuente, Immune cells: free radicals and antioxidants in sepsis. International immunopharmacology, 2004. 4(3): p. 327-347.

52. Arbind, K., R. Sheela Devi, and L. Sundareswaran, Role of antioxidant enzymes in oxidative stress and immune response evaluation of aspartame in blood cells of wistar albino rats. International Food Research Journal, 2014. 21(6): p. 2263-2272.

53. Choudhary, A.K. and R.S. Devi, Exposures of Long Intake of Aspartame on Free Radical Scavenging Enzymes in Blood cells and Neutrophil Functions of immunized wistar albino rats. American Journal of PharmTech Research 2014. 4(1): p. 1014-1029.

54. Ashok, I., R. Sheeladevi, and D. Wankhar, Acute effect of aspartame-induced oxidative stress in Wistar albino rat brain. Journal of biomedical research, 2015. 29(5): p. 390.

55. Abdel-Salam, O.M., N.A. Salem, and J.S. Hussein, Effect of aspartame on oxidative stress and monoamine neurotransmitter levels in lipopolysaccharide-treated mice. Neurotoxicity research, 2012. 21(3): p. 245-255.

56. Knight, J.A., Review: Free radicals, antioxidants, and the immune system. Annals of Clinical & Laboratory Science, 2000. 30(2): p. 145-158.

57. Roy, S., C.K. Sen, and L. Packer, Determination of cell-cell adhesion in response to oxidants and antioxidants. Methods in enzymology, 1999. 300: p. 395-401.

58. Albelda, S.M., C.W. Smith, and P. Ward, Adhesion molecules and inflammatory injury. The FASEB Journal, 1994. 8(8): p. 504-512.

59. Valko, M., et al., Free radicals and antioxidants in normal physiological functions and human disease. The international journal of biochemistry and cell biology, 2007. 39(1): p. 44-84.

60. Bautista, A.P., Neutrophilic infiltration in alcoholic hepatitis. Alcohol, 2002. 27(1): p. 17-21.61. Parthasarathy, N.J., R.S. Kumar, and R.S. Devi, Effect of methanol intoxication on rat neutrophil

functions. Journal of immunotoxicology, 2005. 2(2): p. 115-121.62. Abhilash, M., et al., Effect of long term intake of aspartame on antioxidant defense status in

liver. Food and chemical toxicology, 2011. 49(6): p. 1203-1207.63. Gabay, C., Interleukin-6 and chronic inflammation. Arthritis research & therapy, 2006. 8(2): p. S3.64. Gauldie, J., et al., Interferon beta 2/B-cell stimulatory factor type 2 shares identity with

monocyte-derived hepatocyte-stimulating factor and regulates the major acute phase protein response in liver cells. Proceedings of the National Academy of Sciences, 1987. 84(20): p. 7251-7255.

65. Ley, K., et al., Getting to the site of inflammation: the leukocyte adhesion cascade updated. Nature Reviews Immunology, 2007. 7(9): p. 678.

66. Choudhary, A.K. and R.S. Devi, Effect of long intake of aspartame on oxidative stress and cell and humoral immune response in immunized wistar albino rats. American Journal of PharmTech Research 2014. 4(2): p. 235-250.

67. Arbind, K.C. and S.D. Rathinasamy, Effects of aspartame on hsp70, bcl-2 and bax expression in immune organs of Wistar albino rats. The Journal of Biomedical Research, 2016. 30(5): p. 427–435.

68. Pandurangan, M., et al., Investigation of role of aspartame on apoptosis process in HeLa cells. Saudi journal of biological sciences, 2016. 23(4): p. 503-506.

69. Villareal, L.M.A., et al., Neurotropic effects of aspartame, stevia and sucralose on memory retention and on the histology of the hippocampus of the ICR mice (Mus musculus). Asian Pacific Journal of Tropical Biomedicine, 2016. 6(2): p. 114-118.

70. Ramsland, P.A., et al., Interference of rheumatoid factor activity by aspartame, a dipeptide methyl ester. Journal of Molecular Recognition, 1999. 12(4): p. 249-257.

71. Monte, W.C., Aspartame: methanol and the public health. Journal of Applied Nutrition 1984. 36: p. 42-54.

23

72. Stegink, F., Aspartame: physiology and biochemistry. Vol. 12. 1984: CRC Press.73. Stegink, L.D., et al., Blood methanol concentrations in normal adult subjects administered abuse

doses of aspartame. Journal of Toxicology and Environmental Health, Part A Current Issues, 1981. 7(2): p. 281-290.

74. Kulkarni, K. and M. Franz, Is aspartame safe? The Diabetes Educator, 1984. 10(2): p. 56-57.75. Trocho, C., et al., Formaldehyde derived from dietary aspartame binds to tissue components in

vivo. Life sciences, 1998. 63(5): p. 337-349.76. Makar, A. and T. Tephly, Methanol poisoning in the folate-deficient rat. Nature 1976. 261: p. 715

- 716.77. Black, K.A., et al., Role of hepatic tetrahydrofolate in the species difference in methanol toxicity.

Proceedings of the National Academy of Sciences, 1985. 82(11): p. 3854-3858.78. Maker, A. and T. Tephly, Methanol in poisoning folate deficient rats. Nature, 1976. 261: p. 715-

716.79. Tephly, T.R., The toxicity of methanol. Life sciences, 1991. 48(11): p. 1031-1041.80. Ranney, R., et al., Comparative metabolism of aspartame in experimental animals and humans.

Journal of Toxicology and Environmental Health, Part A Current Issues, 1976. 2(2): p. 441-451.81. Röe, O. and P. Enoksson, Species differences in methanol poisoning. CRC Critical Reviews in

Toxicology, 1982. 10(4): p. 275-286.82. Roberts, H., Aspartame as a cause of allergic reactions, including anaphylaxis. Archives of

internal medicine, 1996. 156(9): p. 1027-1027.83. Roberts, H.J., Reactions attributed to aspartame-containing products: 551 cases. Journal of

Applied Nutrition, 1988. 40: p. 85-94.84. Roberts, H.J., Sweet'ner Dearest: Bittersweet Vignettes About Aspartame (NutraSweet). 1992:

Sunshine Sentinel Press.85. Hill, A.M. and D.V. Belsito, Systemic contact dermatitis of the eyelids caused by formaldehyde

derived from aspartame? Contact dermatitis, 2003. 49(5): p. 258-259.86. Jacob, S.E. and S. Stechschulte, Formaldehyde, aspartame, and migraines: a possible connection.

Dermatitis, 2008. 19(3): p. E10.87. Abegaz, E.G. and R.G. Bursey, Formaldehyde, aspartame, migraines: a possible connection.

Dermatitis, 2009. 20(3): p. 176-177.88. Thrasher, J.D., A. Broughton, and R. Madison, Immune activation and autoantibodies in humans

with long-term inhalation exposure to formaldehyde. Archives of Environmental Health: An International Journal, 1990. 45(4): p. 217-223.

89. Stegink, L.D., et al., Blood methanol concentrations in one-year-old infants administered graded doses of aspartame. The Journal of nutrition, 1983. 113(8): p. 1600-1606.

90. Davoli, E., et al., Serum methanol concentrations in rats and in men after a single dose of aspartame. Food and chemical toxicology, 1986. 24(3): p. 187-189.

91. Stegink, L.D., et al., Effect of repeated ingestion of aspartame-sweetened beverage on plasma amino acid, blood methanol, and blood formate concentrations in normal adults. Metabolism, 1989. 38(4): p. 357-363.

92. Oyama, Y., et al., Cytotoxic effects of methanol, formaldehyde, and formate on dissociated rat thymocytes: a possibility of aspartame toxicity. Cell biology and toxicology, 2002. 18(1): p. 43-50.

24