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Home , Adenosine triphosphate, Aspartame

Special Article Revisiting the safety of

Arbind Kumar Choudhary and Etheresia Pretorius

Aspartame is a synthetic artificial sweetener, frequently used in , medications, and beverages, notably carbonated and powdered soft drinks. Since 1981, when aspartame was first approved by the US and Drug Administration, researchers have debated both its recommended safe dosage (40 mg/kg/d) and its general safety to organ systems. This review examines papers published between 2000 and 2016 on both the safe dosage and higher-than- recommended dosages and presents a concise synthesis of current trends. Data on the safe aspartame dosage are controversial, and the literature suggests there are potential side effects associated with aspartame consumption. Since aspartame consumption is on the rise, the safety of this sweetener should be revisited. Most of the literature available on the safety of aspartame is included in this review. Safety studies are based primarily on animal models, as data from human studies are lim- ited. The existing animal studies and the limited human studies suggest that aspar- tame and its metabolites, whether consumed in quantities significantly higher than the recommended safe dosage or within recommended safe levels, may disrupt the oxidant/ balance, induce , and damage membrane integrity, potentially affecting a variety of cells and tissues and causing a deregula- tion of cellular function, ultimately leading to systemic inflammation.

INTRODUCTION .5 It was first marketed as NutraSweet and and is now freely available in supermarkets. Aspartame Non-nutritive sweeteners are high-intensity sweeteners is incorporated into more than 6000 products, includ- that are used in small amounts to reduce the caloric and ing soft drinks, dessert mixes, frozen desserts and yo- content of food and beverages.1 The controversial in- gurt, chewable multivitamins, and breakfast cereals. It is creased use of non-nutritive sweeteners in so-called healthy also contained in about 600 pharmaceutical products6–9 food and beverages has recently come under the spotlight. and is, therefore, consumed by millions of people In particular, aspartame, which was accidentally discovered worldwide.10 Aspartame metabolites may reduce or po- in 1969,2 has received much attention because of its potent tentiate drug action through various mechanisms.11 , which is 200 to 300 times greater than that of Metabolites amino , and may (1) alter .3 Aspartame has a clean sugar-like , with no blood proteins to which drugs attach; (2) alter drug undesirable metallic or bitter taste. It is far cheaper than receptors on cell membranes; (3) change the sites at sugar and is an attractive option for manufacturers.4 which impulses are transmitted along nerves to muscle; Aspartame is a synthetic dipeptide formed by the (4) cause metabolic abnormalities in elderly people that reaction of L-aspartic with L- methyl may enhance the vulnerability of this population to

Affiliation: A.K. Choudhary is with the Department of Physiology, School of Medicine, Faculty of Health Sciences, University of Pretoria, Pretoria, South Africa. E. Pretorius is with the Department of Physiological Sciences, Faculty of Health Sciences, Stellenbosch University, Stellenbosch, South Africa. Correspondence: E. Pretorius, Department of Physiological Sciences, Faculty of Health Sciences, Stellenbosch University, Private Bag X1 Maiteland, 7602, South Africa. Email: [email protected]. Key words: aspartame, , , phenylalanine.

VC The Author(s) 2017. Published by Oxford University Press on behalf of the International Sciences Institute. All rights reserved. For Permissions, please e-mail: [email protected].

doi: 10.1093/nutrit/nux035 718 ReviewsVR Vol. 75(9):718–730 drug reactions; or (5) interfere with drug action. Safety issues associated with the use of aspartame include po- tential toxicity from aspartame metabolites, including methanol and/or its metabolite, .12,13 This review examines the existing literature (pub- lished 2000–2016) describing the effects of aspartame on cells and organ systems when used within the safe dosage range. The interactions between aspartame and cells and organ systems are examined, and extensive references for current recommended safe dosages are provided. Finally, literature that considers the effects of aspartame on different cells in the body is reviewed. Figure 1 Structure of aspartame (L-aspartyl-L-phenylalanine of aspartame methyl ester).

Aspartame is an L-aspartyl-L-phenylalanine methyl es- further metabolized in the into L-tyrosine by the en- 14 ter (Figure 1) and is very stable under dry conditions zyme phenylalanine hydroxylase. L-Tyrosine in turn, is when stored at temperatures ranging from 30 Cto converted into L-dopa (L-3,4-dihydroxyphenylalanine) by 15 23 80 C. It degrades at high temperatures and in aqueous the tyrosine hydroxylase. L-Dopa is further solutions. The rate of degradation in aqueous solutions, converted into the —, norepi- 16 however, depends on pH as well as temperature. At nephrine (noradrenaline), and epinephrine (adrena- room temperature, aspartame is most stable at pH 4.3, line)—by the decarboxylase enzyme. with a half-life of 300 days. Degradation is minimal Phenylalanine can cross the blood–brain barrier.24,25 when pH ranges between 4.0 and 5.0 and reaches a maxi- Furthermore, elevations in plasma of phe- mum when heated under conditions of high humidity at nylalanine and aspartic acid result in increased transport 16 a pH greater than 6.0. Under strongly acidic of these amino acids into the brain, modifying the brain’s (pH < 4.0) or alkaline conditions (pH > 6.0), aspartame neurochemical composition.26 Neuroendocrine changes, may generate methanol by . Under more se- particularly increased concentrations of vere conditions, such as elevated temperature or high 17 resulting from phenylalanine and its hydroxylation prod- pH, the bonds are also hydrolyzed. This results uct, tyrosine, have been observed in the brain.26 in the release of free amino acids (particularly phenylala- Phenylalanine is a large neutral amino acid that nine and aspartic acid). It should also be noted that the competes with other important large neutral amino pH of sodas—a major vehicle for aspartame acids for binding on the large neutral amino acid trans- consumption—tends to be somewhere between 3.0 and porter.26 However, excess phenylalanine concentrations 4.0. Interestingly, following breakdown in the gut or ex- are associated with decreased concentrations of cate- posure to temperature changes, aspartame and its metab- cholamine, , and dopamine.26 Aspartic acid is olites lose their sweetness.18,19 Conditions during storage metabolized in the liver into L- and L-methionine vs during ingestion are thus different and may determine 20 by the enzyme aspartate . At high concentrations, the formation of aspartame metabolites. aspartic acid may cross the blood–brain barrier and bind to the N-methyl-D-aspartate (also known Aspartame during storage. Aspartame can be stored in a 27 dry or an aqueous form. In the dry form, stability as the NMDA receptor or NMDAR) or to other gluta- depends mainly on temperature, and stability decreases mate binding sites, causing an influx of at temperatures < 30oCor> 80oC. In the aqueous into cells (Figure 2). Increased firing of action potentials form, stability is greatest at a pH of 4.3; beyond this pH, and higher rates of depolarization can potentiate 26 aspartame degrades into its 3 known metabolites (phe- neurodegeneration. The enzyme responsible for me- 28 nylalanine, aspartic acid, and methanol) and loses some tabolism of methanol (Ch3OH) is species dependant. of its sweetness. The aqueous form also becomes In primates, methanol is metabolized into formaldehyde 29 sweeter with increased temperature.21 (HCHO) in the liver by dehydrogenase. In rodents, on the other hand, methanol is mainly metabo- Aspartame during ingestion. Upon ingestion, aspartame lized by alcohol catalase and differences in the embry- is metabolized by gut (esterase and peptidase) onic of CH3OH may determine species into 3 amino acid isolates, phenylalanine (50%), aspartic sensitivity, in which mouse embryos were more sensitive acid (40%), and methanol (10%).22 Phenylalanine is than the rat.30 Formaldehyde is oxidized into formic

Nutrition ReviewsVR Vol. 75(9):718–730 719 Table 1 Phenylalanine and aspartic acid content of as- partame-sweetened beverages and other common foods and beverages Food or beverage Phenylalanine (g) Aspartic acid (g) Aspartame-sweetened 1.186 0.983 beverages (per 100 g) -free or skim milk 0.175 0.288 (per 100 g) Apple (per 100 g) – – Tomato juice (per 100 g) 0.026 0.130 Orange juice (per 100 g) 0.043 0.437 Banana (raw) 0.049 0.124 Egg white (raw) 0.686 1.220

metabolism. The so-called free forms of amino acids found in aspartame-sweetened beverages are released Figure 2 Binding of aspartate to NMDA (N-methyl-D-aspartate) receptor structures. The NMDA receptor has 3 binding sites: a rapidly and in greater concentrations. For example, the glutamate , a binding site, and an allosteric methanol released during aspartame metabolism is a binding site. When aspartate binds to the glutamate-binding site, free form and is thus released directly into the blood- it results in the opening of the Ca21 channel and allows Ca21 stream.45 It was also recently shown that, after an ac- into cells, leading to a higher rate of neuron depolarization. ceptable daily intake (ADI) of 40 mg per kilogram of body weight, blood methanol concentrations increased acid (HCOOH) by formaldehyde dehydrogenase in both 3- to 6-fold over individual baseline values.46 primates and rodents. is metabolized more The daily consumption of chemically produced as- rapidly into dioxide and in rodents,31 as partame is increasing. Understanding the adverse rodents produce more folic acid (tetrahydrofolate) than effects of aspartame and its metabolites is thus essential. primates. Excess formic acid may lead to metabolic aci- The safe levels of non-nutritive artificial sweeteners, es- dosis and tissue injury, with humans being uniquely sen- pecially aspartame in soft drinks, iced tea, concentrated sitive because of their low hepatic folate concentrations.32 fruit , fruit and vegetable , flavored water, drinks, milk-based desserts, candies, jelly, Safety of aspartame consumption chewing gum, fruit yogurt, and are continu- ously monitored and scrutinized by governmental agen- The safety of aspartame and its metabolites (phenylala- cies. The European Authority set the ADI nine, aspartic acid, and methanol) has been discussed of aspartame for humans at 40 mg/kg of body weight.47 frequently,33–36 and blood concentrations of aspartame The US Food and Drug Administration set an ADI of metabolites increase after consumption.37–39 An impor- 50 mg/kg of body weight.48 The ADI of aspartame can tant consideration is that aspartame metabolites are also be calculated as follows: found naturally in foods (Table 140). Milk has approximately 6 times more phenylala- ðmg=ðÞ¼kg ðÞaspartame ½lg=g daily consumption nine and 13 times more aspartic acid than the same vol- ðÞg=person=d body weightðÞ kg ume of an aspartame-sweetened beverage.41 Similarly, Daily consumption of artificial sweeteners by tomato juice has 6 times more methanol than an equiv- 41 women of childbearing age and by children has been es- alent volume of an aspartame-sweetened beverage. timated at 2.5 to 5.0 mg/kg.34 Typically, in adults, mean Exposure to low levels of methanol, which occurs natu- intake values of aspartame range from 5.6% of the ADI rally in blood, urine, saliva, and expired air, is com- to, at most, 14.7% of the ADI. Mean intake values in 42 mon. Methanol occurs in fresh fruits and juices, children range from 21% of the ADI to, at most, 43.1% 43,44 vegetables, and fermented beverages. It is produced of the ADI. endogenously when compounds such as pectin are fer- The ADI of aspartame is also species dependent. 31 mented. Naturally occurring phenylalanine, aspartic Body surface area conversions should be used to convert acid, and methanol are released at different rates to pro- between rat ADIs and human ADIs.49,50 Thesafedoseor duce artificial or free forms of these compounds. ADI of aspartame for humans, ie, 40 mg/kg, is always cor- Naturally occurring amino acids like aspartic acid and rected by a factor of 5 or 6 for rats, as rats metabolize as- phenylalanine are bound to a , and thus they are partame faster than humans.51,52 From these calculations, released slowly into the body during digestion and it is accepted that rats may have an aspartame dosage of

720 Nutrition ReviewsVR Vol. 75(9):718–730 250 mg/kg/d (after species factor correction). It is there- induce selective loss of T- molecules. This fore suggested that the oral lethal dose of aspartame in may lead to decreased T-cell proliferation94 and can ul- ratsandmiceismorethan10g/kg/d.53 timately result in .111 Normally, in the coagulation process, thrombin, fi- DISCUSSION brin, and play an important role in hemostasis. An increase in oxidant stress and a decrease in antioxi- Absorption and toxicokinetic data that compare the dant levels are also associated with aberrant changes in effects of aspartame in humans and animals at the same function.112 However, fibrin formation (and, dosages are not available. However, as noted previously, therefore, fibrin packaging) and platelet were animals respond to aspartame and/or aspartame metab- found to be changed during aspartame intake in an ani- olites more rapidly than do humans.54 Extrapolating in- mal model.73 The hydrogen peroxide and peroxyl radi- formation from animal studies is one of the limitations cals that form when aspartame is ingested13,74 are likely of using data from animal studies. This review presents involved in enhanced calcium mobilization, which may information from recent studies (2000–2016) that inves- lead to platelet hyperactivity and hyperaggregability in tigated excess dosages (> 40 mg/kg/d) (Table 237,55–69) patients with type 2 diabetes.95 Type 2 diabetes causes and optimum safe dosages for humans ( 40 mg/kg/d) changes in the coagulation system, and hypercoagul- (Table 312,13,55,70–90). Table 412,75,81,84–87,89–107 summarizes ability is a hallmark of the systemic inflammatory pro- the effects of aspartame administered at higher and at safe file in type 2 diabetic patients. Hence, aspartame use dosages. The effects of aspartame on different cells and or- may exacerbate the hypercoagulability already present gan systems are detailed below, in the remaining sections. in these patients.

Effect of aspartame on blood cells and fibrin Effect of aspartame on the brain packaging Once the cellular antioxidant capacity is overcome by Injury to cell membranes by free radicals can lead to a the generation of ROS and reactive species change in cell membrane fluidity, impairing the vital (RNS), cellular damage may occur.92 Neuronal cells are functions of blood cells (erythrocytes, neutrophils, and especially vulnerable to oxidative stress because of high lymphocytes).92 Most importantly, it can affect immu- concentrations of polyunsaturated fatty acids, which nity by altering neutrophil and lymphocyte function.93 render them much more susceptible to peroxida- Oral administration of aspartame at both a higher dos- tion compared with other tissues.113 The excess free age (> 40 mg/kg/d)55 and a safe dosage ( 40 mg/kg/ radicals can attach to fatty acids in the neuronal cell d)55,70,71 can increase the production of free radicals membrane, thereby interfering with neuronal cell func- and induce oxidative stress in blood cells (erythrocytes, tion.97 The production of excess free radicals may also neutrophils, and lymphocytes) by altering the oxidant/ increase permeability of the blood–brain barrier in a antioxidant balance. Oxidative stress in erythrocytes time- and -dependent manner.98 can lead to damage of the erythrocyte membrane108; The consumption of higher dosages of aspartame impair the flow of erythrocytes through the microcircu- (> 40 mg/kg/d) on the brain was also previously stud- lation and the delivery of to the tissues91; induce ied.56–60,62–64 Results suggest that higher aspartame dos- erythrocyte aging91; and induce inflammation.109 ages may result in changed enzyme activities.56–60,63–64 Reactions between excessive amounts of reactive It was also found, in an in vivo voltammetry study that oxygen species (ROS) and superoxide radicals from ac- aspartame decreases evoked extracellular dopamine lev- tivated neutrophils can exert cytotoxic effects and may els in the rat brain.62 Brain areas affected by aspartame induce overactivation of the nuclear repair enzyme may include the cerebral cortex, hypothalamus and hip- poly-( diphosphate [ADP]-) polymer- pocampus, as shown in a paper by Iyyaswamy and ase, which may cause depletion Rathinasamy in 2012.60 Areas like the hippocampus and and cellular injury.96 The activation of poly-(ADP-ri- medial prefrontal cortex play important roles in mem- bose) polymerase is also known to upregulate multiple ory and decision making.114,115 Even the US Food and pathways of proinflammatory signaling.96 Hence, oxida- Drug Administration–approved ADI of aspartame tive stress in blood cells after aspartame consumption ( 40 mg/kg) has been shown to be toxic to the can modify the expression or activation of inflamma- brain.13,74,116 Inside neuronal cells, intense or prolonged tory mediators. The T lymphocytes (T cells) are ren- oxidative stress causes overexpresssion of proinflamma- dered hyporesponsive to activating stimuli, but both tory cytokines (interleukin 6 [IL-6], interleukin 1b exposure to ROS produced by activated neutrophils110 [IL-1b], interleukin 8 [IL-8]), considered a hallmark of and prolonged exposure to high ROS concentrations94 neuroinflammation.100,101 The proinflammatory cytokines

Nutrition ReviewsVR Vol. 75(9):718–730 721 Table 2 Effects of high dosages of aspartame (> 40 mg/kg) on different cells and organ systems Reference Cells/ or Aspartame Route and Results tissue animal model dosage duration of administration Tsakiris et al. Blood Human eryth- (150 mg/kg or Incubated for Significant decrease in AChE activity in (2006)55 rocyte 200 mg/kg) 1 h at 37C human erythrocyte membranes membrane when incubated with aspartame components Simintzi et al. Brain Suckling (150 or 200 Incubated for Significant decrease in AChE activity in (2007)56,57 (21 days) mg/kg) 1 h at 37C the frontal cortex and hippocampus Wistar rats with aspartame components Ashok et al. (2013),58 Wistar albino 75 mg/kg Oral for 90 d Imbalance of cell membrane homeosta- Ashok et al. male rats sis, leading to oxidative stress in dis- (2014),59 Iyyaswamy crete brain regions (cerebral cortex, & Rathinasamy cerebellum, midbrain, pons medulla, (2012)60 hippocampus, and hypothalamus) and changes in locomotor activity and anxiety levels. Histopathological alterations in brain regions were also observed Villareal et al. Male ICR 1000 mg/kg Oral for 32 d No significant difference on memory (2016)61 mice retention, but a neurotropic effect (Mus (apoptosis) was observed in the musculus) hippocampus (brain) Abhilash et al. Wistar albino 500 mg/kg and Oral for 90 d Imbalance in antioxidant/prooxidant (2013)64 male rats 1000 mg/kg status, mainly through the glutathi- one-dependent system, which led to vascular congestion in the brain Bergstrom et al. Sprague- 500 mg/kg Single systemic Relatively potent effect of decreasing (2007)62 Dawley dose evoked extracellular dopamine (DA) rats levels when administered systemi- cally under the conditions specified by the authors Vences-Mejıa et al. Wistar rats 75 and 125 mg/kg Oral for 30 d Increase in protein expression and ac- (2006)63 tivity of several cytochrome P450 enzymes (CYP1, 2, and 3) of phase I metabolizing enzymes in the brain (cerebrum and cerebellum) Abdel-Salam et al. Adult mice 0.625, 1.875, or Subcutaneous At highest dose of 5.625 mg/kg, re- (2012),65 Abdel- 5.625 mg/kg for 2 wk peated aspartame administration im- Salam et al. paired memory and increased (2012)66 oxidative stress in brain. When a mild systemic inflammatory response was present, intraperitoneal administra- tion of LPS (100 mg/kg) increased oxidative stress and inflammation in the brain, but not in the liver Park et al. Adult mice 0.5 mg/g Intraperitoneal Impaired memory retention observed, (2000)67 as well as damaged in the arcuate nucleus of the hypothalamus Goerss et al. Male Long- 200, 400, Intraperitoneal Inverse relationship observed between (2000)37 Evans or 800 mg/kg striatum serotonin levels and aggres- rats sion at high doses of aspartame Ashok et al. Liver Wistar albino 75 mg/kg Oral for 90 d Altered levels of liver marker enzyme (2014)68 male rats (Ygt) as well as histological changes in the liver observed Abhilash et al. Wistar albino 500 mg/kg and Oral for 90 d Aspartame (1000 mg/kg) led to (2011)69 male rats 1000 mg/kg alterations in liver antioxidant status, mainly through the - dependent system and hepatocellu- lar injury Ashok et al. Kidney Wistar albino 75 mg/kg Oral for 90 d Induced histological changes in the (2014)68 male rats renal cortex (kidney) Abbreviations: AChE, acetylcholinesterase; LPS, lipopolysaccharide.

722 Nutrition ReviewsVR Vol. 75(9):718–730 urto Reviews Nutrition Table 3 Effects of safe dosages of aspartame (£ 40 mg/kg) on different cells and organ systems Reference Cells/tissue In vitro or animal model Aspartame dosage Route and duration Results of administration Choudhary & Devi Blood Wistar albino male rats 40 mg/kg Oral for 15, 30, and 90 d Induced oxidative stress in se- 70 V

R (2014) rum, irrespective of duration o.75(9):718–730 Vol. of exposure Arbind et al. (2014)71 Wistar albino male rats 40 mg/kg Oral for 15, 30, and 90 d Induced oxidative stress in blood cells (RBCs, neutrophils, and lymphocytes). Altered neutro- phil function, irrespective of duration of exposure Agamy (2009)72 Diabetic male Wistar rats 40 mg/kg Intraperitoneal injection Significant increase in AChE ac- for 4 wk tivity in serum Pretorius & Humphries Rabbit 34 mg/kg Oral for 2 mo Disruption in platelet activation (2007)73 and coagulation Tsakiris et al. (2006)55 Human erythrocyte 34 mg/kg Incubated for 1 h at 37C Significant decrease in AChE ac- membrane tivity in the human erythro- cyte membrane when incubated with aspartame components Ashok & Sheeladevi Brain Wistar albino male rats 40 mg/kg Oral for 90 d Alterations in neurobehaviors (2015)13, Ashok & (emotional and anxiety behav- Sheeladevi (2014)74 ior). Increased neuronal oxida- tive damage led to neuronal cell death (apoptosis) in dis- crete regions of brain (eg, ce- rebral cortex, cerebellum, midbrain, pons medulla, hip- pocampus, and hypothalamus) Choudhary & Wistar albino male rats 40 mg/kg Oral for 90 d Alteration in EEG pattern (fronto- Sundareswaran parietal and occipital regions) (2016)75 Abu-Taweel et al. Mice 32 mg/kg Oral for 30 d Alterations in behavioral param- (2014)76 eters (cognitive responses, memory retention, and learn- ing capabilities), without sig- nificant changes in biochemical parameters Kim et al. (2011)77 Zebra fish (10 wk old) 3mM Oral for 12 d Increased brain inflammation, impairment of learning and memory, and acute swimming defects were noted in hyperli- pidemic rats (continued) 723 724 Table 3 Continued Reference Cells/tissue In vitro or animal model Aspartame dosage Route and duration Results of administration Christian et al. (2004)78 Male Sprague–Dawley rats Rat dosage (250 mg/kg) Oral, via the drinking Altered T-maze performance and water, for 3–4 mo increased muscarinic choliner- gic receptor or enzymes in certain brain regions Abd El-Samad (2010)79 Wistar albino male rats Rat dosage (250 mg/kg) Oral for 8 wk Harmful effects (condensed nu- clei and loss of characteristic pyriform shape of Purkinje cells) on cells in the cerebellar cortex Ashok & Sheeladevi Liver Wistar albino male rats 40 mg/kg Oral for 90 d Altered antioxidant status, ex- (2015)80 pression of stress protein, and induced apoptotic changes in the liver Choudhary & Devi Wistar albino male rats 40 mg/kg Oral for 15 d and 30 d Induced oxidative stress in liver, (2014),70 Kumar regardless of duration of ex- Choudhary et al. posure. Levels of serum pro- (2014)81 tein and bilirubin that reflect liver function were altered af- ter 30 d of aspartame administration Iman (2011)82 Wistar albino male rats 40 mg/kg Oral for 2, 4, and 6 wk Induced oxidative stress in liver after 4 wk and 6 wk of treatment Kim et al. (2011)77 Zebrafish (10 wk old) 3mM Oral for 12 d Inflammatory cells in the liver were infiltrated Choudhary & Devi Kidney Wistar albino male rats 40 mg/kg Oral for 15 d and 30 d Induced oxidative stress in kid- (2014),70 Kumar ney and serum values that re- Choudhary et al. flect kidney function (such as (2014)81 , urea, and ) after 30 d of aspartame administration urto Reviews Nutrition Iman (2011)82 Wistar albino male rats 40 mg/kg Oral for 2, 4, and 6 wk Induced oxidative stress in kid- ney after 6 wk Martins & Azoubel Wistar female rats (14 mg/kg) heated Intragastric on days 9, 10, Morphometric alterations in all (2007)83 to 40C and 11 of pregnancy renal structures (glomerulus, proximal and distal convo- V R luted tubules, and collecting o.75(9):718–730 Vol. ducts) of the rat fetal kidney during organogenesis (continued) urto Reviews Nutrition Table 3 Continued Reference Cells/tissue In vitro or animal model Aspartame dosage Route and duration Results of administration Choudhary & Heart Wistar albino male rats 40 mg/kg Oral for 90 d Induced oxidative stress in the Sundareswaran heart, impaired cardiac func- V R 75 o.75(9):718–730 Vol. (2016), Choudhary & tion, and reduced heart rate Sundareswaran variability, as evidenced by (2016)84 sympathetic dominance and loss of vagal tone, but could not induce structural change Gudadhe et al. (2013)85 Neonatal mice 100 mg/g Intraperitoneal for 2 wk Compensatory hypertrophy of myocytes as a toxic effect of aspartame Choudhary & Devi Immune system Wistar albino male rats 40 mg/kg Oral for 90 d Altered the homeostasis of im- (2014),12 Kumar mune organs. Possible oxida- Choudhary et al. tive stress and imbalanced (2014),81 Choudhary & oxidant/antioxidant status, Devi (2015),86 variations in serum cytokine Choudhary & levels, and alteration of cellu- Rathinasamy (2014)87 lar and humoral immunity Okasha (2016)88 Sciatic nerve Male albino rats Rat dosage (250 mg/kg) Oral for 3 mo Degenerative changes observed, mainly in the myelin sheath in the form of focal and exten- sive demyelination Palmn€as et al. (2014)89 Gut microbes Male albino rats 5–7 mg/kg Oral for 8 wk Elevated fasting levels and impaired tolerance may both be mediated by al- teration of Suez et al. (2014)90 Mice 4% aspartame Oral for 11 wk Induced higher glucose excur- sions, mediated by alteration of gut microbiota Abbreviations: AChE, acetylcholinesterase; EEG, electroencephalogram; RBCs, red blood cells. 725 Table 4 Effects of aspartame (higher and safe dosages) on different cells and organ systems Cells/tissue At higher dosages At safe dosages Effects (> 40 mg/kg) ( 40 mg/kg) Blood Affected Affected Impaired delivery of oxygen to the tissues by red blood cells91,92; aging91; altered neutrophil function93; decreased T-cell prolifera- tion94; platelet hyperactivity and hyperaggreg- ability95; upregulation of proinflammatory signaling96 Brain Affected Affected Dysfunction of neuronal cells97; disruption of blood–brain barrier98; impaired neurobehavio- ral parameters (learning and memory)99; upre- gulation of neuroinflammation, which may initiate neurotropic effects100,101 Liver Affected Affected Loss of liver function102; inactivation of the group103,104; defect in synthesis of clotting factor105 Kidney Affected Affected Loss of kidney function106,107; weakening of acid– balance106 Heart Not measured Affected Impaired cardiac function84; reduced heart rate variability75; and compensatory hypertrophy of myocytes85 caused by oxidative stress Immune organs Not measured Affected Imbalanced oxidant/antioxidant status and varia- tions in serum cytokine levels, eventually result- ing in alteration of cellular and humoral immunity12,81,86,87 Gut microbes Not measured Affected Elevated fasting glucose and impaired insulin tol- erance, mediated by alteration of the gut microbiota89,90 Lungs, pancreas, Not measured Not measured Unknown endocrine glands, reproductive organs

are important regulators of matrix metalloproteinases,98 regenerative processes,121 resulting in hepatic dam- which are zinc-containing enzymes produced by acti- age.102 The cytotoxic effects of ROS and RNS in the vated microglia. They are responsible for breaking down liver may also lead to inactivation of the heme group the extracellular matrix in cerebral blood vessels, which and nitrosylation of – group.103,104 In addi- leadstodamageofneurons117 and a disruption in the tion, the inflammation caused by the presence of ROS blood–brain barrier.118 results in the modulation of hepatocyte metabolism.105 Neurobehavioral parameters (like learning and Furthermore, Kupffer cells activated by free radicals are memory) were impaired not only with higher dosages responsible for the release of proinflammatory cyto- of aspartame65,67 but also with safe dosages.75–78 At kines.122 The toxicity of methanol released during the high concentrations, ROS and RNS may lead to de- metabolism of aspartame was also shown to lead to apo- creased by attenuating long-term po- ptotic changes in the liver of Wistar albino rats.80 tentiation and synaptic .99 Effect of aspartame on the kidney Effect of aspartame on the liver The kidneys are vital organs, necessary for maintaining The liver is the principal detoxifying organ and main- the composition and volume of body fluids, the acid– tains metabolic homeostasis.119 Free radical reactions base balance, and the status.106 Oxidative stress are widely known to be involved in liver injury.120 Both may lead to kidney injury.107 Both higher dosages and higher dosages of aspartame68,69 and safe dos- safe dosages of aspartame may induce oxidative stress ages70,77,81,82 have been shown to impair the antioxidant in the kidneys of rats.68,70,81–83 It was previously sug- status of the liver, which may lead to hepatocellular in- gested that in the kidney, oxidative stress may contrib- jury. During oxidative stress, the balance between ROS ute to the progression of kidney fibrosis,123 and chronic and shifts toward increased production kidney disease may be associated with inflammation of the former, which may affect the energetic and and upregulated inflammatory cytokines.124

726 Nutrition ReviewsVR Vol. 75(9):718–730 Effect of aspartame on the heart (including cardiometabolic effects)

Over the past decade, a number of animal studies—as well as multiple large-scale, long-term, prospective ob- servational studies in humans—have reported increased incidences of a range of cardiometabolic conditions among study participants with daily dietary exposure to aspartame. Fowler125 conducted an extensive review of results from animal research as well as from large-scale, long-term observational studies in humans on the car- diometabolic risks of aspartame use. Myocardial func- tion is also modulated by the .126 Aspartame may lead to oxidative stress in cardiac tissue and has been shown to impair cardiac Figure 3 Role of aspartame in the development of inflamma- 84 function, resulting in reduced heart rate variability, tion. Consumption of aspartame may lead to excess production of sympathetic dominance, and loss of vagal tone.75 The ROS and RNS, inducing oxidative stress in different cells and tis- loss of protective vagal tone would explain the increased sues. This causes dysregulation of proinflammatory markers, which susceptibility to cardiovascular disease.126 An increasing ultimately leads to chronic systemic inflammation, which in turn causes gut bacterial dysregulation. Abbreviations: CRP, C-reactive number of physical illnesses appear to be associated protein; GIT, gastrointestinal tract; iNOS, inducible syn- with sympathetic dominance, reduced vagal tone, and thase; RNC, reactive nitrogen species; ROS, . reduced heart rate variability.127 The inflammatory markers fibrinogen and IL-6 are both moderately re- Effect of aspartame on the gut microbiota lated to heart rate variability, which demonstrates a re- lationship between autonomic nervous system function Non-nutritive sweeteners (including aspartame) may and inflammatory and coagulant processes.128 This sug- influence gut metabolism by changing the host meta- gests that oxidative stress or inflammation caused by as- bolic phenotype, ultimately affecting the gut micro- 131 partame could affect heart rate variability. The toxic biota. Changes in the gut microbiota may interfere effect of aspartame may also induce structural changes with the physiological responses that control homeosta- in the myocardium that manifest as compensatory hy- sis; alter the intestinal environment, thereby triggering pertrophy of myocytes.85 inflammatory processes associated with metabolic dis- orders; or disrupt sweet-taste receptors in the gut that Effect of aspartame on the immune system can affect glucose absorptive capacity and glucose homeostasis. The oxidant/antioxidant balance is critical for immune Aspartame, which has bacteriostatic properties, cell function, since it maintains the integrity and func- also has an anticavity effect and is resistant to fermenta- tionality of cellular proteins, nucleic acids, and the cell tion by oral bacteria.132 Aspartame’s ability to curb the membrane.93 Immune cells are particularly sensitive to growth of bacteria is not limited to oral bacteria but oxidative stress because of the high percentage of poly- extends to the gut microbiota in animal models. Low- unsaturated fatty acids in their plasma membranes.129 dose aspartame (5–7 mg/kg/d) consumed in drinking Aspartame (40 mg/kg/d) may act as a chemical stressor water over an 8-week period resulted in elevated fasting and induce oxidative stress by disturbing the oxidant/ glucose levels and impaired insulin tolerance in diet- antioxidant balance,12 which may alter the ability of the induced obese rats.89 Mice that drank water containing immune system to maintain homeostasis, as observed 4% aspartame and ate a high-fat diet for 11 weeks had in an animal model in which both nonimmunized87 higher glucose excursions after a glucose load; the elimi- and immunized81 rats were exposed to aspartame. This nation of this effect by treatment suggested oxidant/antioxidant imbalance may lead to variations in that changes in the metabolic phenotype of the mice serum cytokine levels and to alterations in both cellular was caused by alterations in the gut microbiota.90 and humoral immunity.86 The release of ROS has long been recognized as a typical consequence of immune CONCLUSION cell stimulation,130 but excess production of ROS can promote inflammation by direct oxidative damage or Current scientific knowledge about the safety of aspar- by alteration of innate and adaptive mechanisms.130 tame, as reviewed here, is based mostly on animal

Nutrition ReviewsVR Vol. 75(9):718–730 727 studies. These studies suggest that aspartame, even at 11. Roberts HJ. Aspartame disease: an FDA-approved epidemic. http://articles.mer cola.com/sites/articles/archive/2004/01/07/aspartame-disease-part-two.aspx. recommended safe dosages, might not be safe. Several Published January 7, 2004. Accessed July 24, 2017. of these studies (in vitro as well as in vivo) that investi- 12. Choudhary AK, Devi RS. Imbalance of the oxidant–antioxidant status by aspar- tame in the organs of immune system of Wistar albino rats. Afr J Pharm gated both higher and safe dosages indicate that aspar- Pharmacol. 2014;8:220–230. tame or its metabolites cause an oxidant/antioxidant 13. Ashok I, Sheeladevi R. Neurobehavioral changes and activation of neurodegener- ative apoptosis on long-term consumption of aspartame in the rat brain. J Nutr imbalance, induce oxidative stress, and damage mem- Intermed Metabol. 2015;2:76–85. brane integrity (lipid, protein, and ), possi- 14. Nakanishi K, Kamikubo T, Matsuno R. Continuous synthesis of N-(benzyloxycar- bonyl)-L-aspartyl-L-phenylalanine methyl ester with immobilized in bly affecting most cells and tissues. Aspartame is an organic solvent. Nat Biotechnol. 1985;3:459–464. directly involved in the development of oxidative stress, 15. Bell LN, Labuza TP. Aspartame degradation kinetics as affected by pH in interme- diate and low moisture food systems. J Food Sci. 1991;56:17–20. which is a hallmark of systemic inflammation (Figure 3). 16. Sun J-P, Han Q, Zhang X-Q, et al. Investigations on the degradation of aspartame Several animal studies have also reported a deleterious using high-performance liquid chromatography/tandem mass spectrometry. Chin Chem Lett. 2014;25:1259–1264. effect of aspartame exposure on body weight, adiposity, 17. Chattopadhyay S, Raychaudhuri U, Chakraborty R. Artificial sweeteners—a re- and/or glucose tolerance and insulin levels. These are view. J Food Sci Technol. 2014;51:611–621. 125 18. Quinlan M, Mialon V, Everitt M. Effect of storage on the flavours of cola drinks summarized in a 2016 review by Fowler. Thus, there sweetened with different sweetener systems. In: Corti, ed: Low-Calorie is a need for additional detailed human studies and Sweeteners: Present and Future. Basel, Switzerland: Karger Publishers; comprehensive characterizations of the physiological 1999:58–63. 19. Wetzel CR, Bell LN. Chemical stability of encapsulated aspartame in cakes with- processes affected by aspartame. This is of particular out added sugar. Food Chem. 1998;63:33–37. importance, as diabetic and other individuals with gut 20. Kroger M, Meister K, Kava R. Low-calorie sweeteners and other sugar substitutes: a review of the safety issues. Compr Rev Food Sci Food Saf. 2006;5:35–47. dysbiosis may already be at increased risk of systemic 21. Paix~ao JA, Rodrigues JB, Esmerino EA, et al. Influence of temperature and fat inflammation because of the inflammatory nature of content on ideal sucrose concentration, sweetening power, and sweetness equivalence of different sweeteners in chocolate milk beverage. J Dairy Sci. their conditions. Data reviewed in this paper suggest 2014;97:7344–7353. that aspartame use could not only exacerbate existing 22. Singh M, Kumar A, Tarannum N. Water-compatible ‘aspartame’-imprinted poly- mer grafted on silica surface for selective recognition in . Anal systemic inflammation but also cause inflammation if Bioanal Chem. 2013;405:4245–4252. healthy individuals ingest it on a regular basis. 23. Daubner SC, Melendez J, Fitzpatrick PF. Reversing the substrate specificities of phenylalanine and tyrosine hydroxylase: aspartate 425 of tyrosine hydroxylase is essential for L-DOPA formation. Biochemistry. 2000;39:9652–9661. Acknowledgments 24. Choi TB, Pardridge WM. Phenylalanine transport at the human blood-brain bar- rier. Studies with isolated capillaries. J Biol Chem. 1986;261:6536–6541. Funding/support. E.P. received grants from the National 25. Weglage J, Wiedermann D, Denecke J, et al. Individual blood–brain barrier phe- nylalanine transport determines clinical outcome in . Ann Research Foundation of South Africa (Competitive Neurol. 2001;50:463–467. Program: 91548) and the Medical Research Council of 26. Humphries P, Pretorius E, Naude H. Direct and indirect cellular effects of aspar- tame on the brain. Eur J Clin Nutr. 2008;62:451–462. South Africa (Self-Initiated Research Program). 27. McBain C, Mayer M. N-methyl- D-aspartic acid receptor structure and function. Physiol Rev. 1994;74:723–760. 28. Oppermann JA. Aspartame metabolism in animals. In: Stegink LD, Filer Jr LJ, eds. Declaration of interest. The authors have no relevant Aspartame: Physiology and Biochemistry. New York, NY: Marcel Dekker. interests to declare. 1984:141–159. 29. Cederbaum AI. Alcohol metabolism. Clin Liver Dis. 2012;16:667–685. 30. Hansen JM, Contreras KM, Harris C. Methanol, formaldehyde, and sodium for- mate exposure in rat and mouse conceptuses: a potential role of the visceral yolk sac in embryotoxicity. Birth Defects Res A Clin Mol Teratol. 2005;73:72–82. REFERENCES 31. Barceloux D, Bond G, Krenzelok E, et al. American Academy of Clinical Toxicology practice guidelines on the treatment of methanol . J Toxicol Clin 1. Figlewicz D, Ioannou G, Jay JB, et al. Effect of moderate intake of sweeteners on Toxicol. 2002;40:415–446. metabolic health in the rat. Physiol Behav. 2009;98:618–624. 32. Johlin FC, Fortman CS, Nghiem D, et al. Studies on the role of folic acid and 2. Mazur RH, Schlatter JM, Goldkamp AH. Structure-taste relationships of some folate-dependent enzymes in human methanol poisoning. Mol Pharmacol. . J Am Chem Soc. 1969;91:2684–2691. 1987;31:557–561. 3. Marinovich M, Galli CL, Bosetti C, et al. Aspartame, low-calorie sweeteners and 33. Magnuson B, Burdock G, Doull J, et al. Aspartame: a safety evaluation based on disease: regulatory safety and epidemiological issues. Food Chem Toxicol. current use levels, regulations, and toxicological and epidemiological studies. 2013;60:109–115. Crit Rev Toxicol. 2007;37:629–727. 4. Nofre C, Tinti J-M. : discovery, properties, utility. Food Chem. 34. Butchko HH, Stargel WW, Comer CP, et al. Aspartame: review of safety. Regul 2000;69:245–257. Toxicol Pharmacol. 2002;35(2 pt 2):S1–S93. 5. Rangan C, Barceloux DG. Food additives and sensitivities. Dis Mon. 35. Soffritti M, Padovani M, Tibaldi E, et al. The carcinogenic effects of aspartame: 2009;55:292–311. the urgent need for regulatory re-evaluation. Am J Ind Med. 2014;57:383–397. 6. Rencu¨zogulları E, Tu¨ylu¨ BA, Topaktas¸ M, et al. Genotoxicity of aspartame. Drug 36. Yılmaz S, Uc¸ar A. A review of the genotoxic and carcinogenic effects of aspar- Chem Toxicol. 2004;27:257–268. tame: does it safe or not? Cytotechnology. 2014;66:875–881. 7. Alleva R, Borghi B, Santarelli L, et al. In vitro effect of aspartame in angiogenesis 37. Goerss AL, Wagner GC, Hill WL. Acute effects of aspartame on aggression and induction. Toxicol in Vitro. 2011;25:286–293. neurochemistry of rats. Life Sci. 2000;67:1325–1329. 8. Duggett A. Medications that contain aspartame. Drugsdb.com website. http:// 38. Dailey J, Lasley S, Burger R, et al. Amino acids, monoamines and audiogenic seiz- www.drugsdb.com/cib/aspartame/medications-that-contain-aspartame/. ures in genetically epilepsy-prone rats: effects of aspartame. Epilepsy Res. Published June 11, 2012. Accessed January 5, 2017. 1991;8:122–133. 9. Drug products containing phenylalanine. National PKU News website. https:// 39. Stegink LD, Filer L, Bell EF, et al. Repeated ingestion of aspartame-sweetened pkunews.org/drug-products-containing-phenylalanine/. Updated 2007. Accessed beverages: further observations in individuals heterozygous for phenylketonuria. January 5, 2017. Metabolism. 1990;39:1076–1081. 10. Butchko HH, Stargel WW. Aspartame: scientific evaluation in the postmarketing 40. USDA Nutrient Data Laboratory, Food and Nutrition Information Center, and period. Regul Toxicol Pharmacol. 2001;34:221–233. Information Systems Division of the National Agricultural Library. USDA Food

728 Nutrition ReviewsVR Vol. 75(9):718–730 Compostion Databases. https://ndb.nal.usda.gov/ndb/. Revised May 2016. 68. Ashok I, Wankhar D, Sheeladevi R, et al. Long-term effect of aspartame on the Accessed July 17, 2017. liver antioxidant status and histopathology in Wistar albino rats. 41. Butchko HH, Kotsonis FN. vs actual intake: the aspartame and Preventive Nutrition. 2014;4:299–305. example. J Am Coll Nutr. 1991;10:258–266. 69. Abhilash M, Paul MS, Varghese MV, et al. Effect of long term intake of aspartame 42. Ernstga˚rd L, Shibata E, Johanson G. Uptake and disposition of inhaled methanol on antioxidant defense status in liver. Food Chem Toxicol. 2011;49:1203–1207. vapor in humans. Toxicol Sci. 2005;88:30–38. 70. Choudhary AK, Devi RS. Serum biochemical responses under oxidative stress of 43. Hou C-Y, Lin Y-S, Wang YT, et al. Effect of storage conditions on methanol con- aspartame in wistar albino rats. Asian Pac J Trop Dis. 2014;4(suppl 1):S403–S410. tent of fruit and vegetable juices. J Food Compost Anal. 2008;21:410–415. 71. Arbind K, Sheela Devi R, Sundareswaran L. Role of antioxidant enzymes in oxida- 44. Dorokhov YL, Shindyapina AV, Sheshukova EV, et al. Metabolic methanol: molec- tive stress and immune response evaluation of aspartame in blood cells of wistar ular pathways and physiological roles. Physiol Rev. 2015;95:603–644. albino rats. Int Food Res J. 2014;21:2263–2272. 45. Davoli E, Cappellini L, Airoldi L, et al. Serum methanol concentrations in rats and 72. Agamy N. Effects of the natural sweetener () and the artificial sweetener in men after a single dose of aspartame. Food Chem Toxicol. 1986;24:187–189. (aspartame) on some biochemical parameters in normal and alloxan-induced di- 46. Spanel P, Dryahina K, Vicherkova P, et al. Increase of methanol in exhaled breath abetic rats. New Biotechnol. 2009;25:S12. doi:10.1016/j.nbt.2009.06.032 quantified by SIFT-MS following aspartame ingestion. J Breath Res. 73. Pretorius E, Humphries P. Ultrastructural changes to rabbit fibrin and platelets 2015;9:047104. doi:10.1088/1752-7155/9/4/047104 due to aspartame. Ultrastruct Pathol. 2007;31:77–83. 47. Renwick A, Nordmann H. First European conference on aspartame: putting safety 74. Ashok I, Sheeladevi R. Biochemical responses and mitochondrial mediated acti- and benefits into perspective. Synopsis of presentations and conclusions. Food vation of apoptosis on long-term effect of aspartame in rat brain. Redox Biol. Chem Toxicol. 2007;45:1308–1313. 2014;2:820–831. 48. Di Pasquale M. Use of nutritive and nonnutritive sweeteners. J Am Diet Assoc. 75. Choudhary AK, Sundareswaran L, Devi RS. Effects of aspartame on the evaluation 1998;98:580–587. of electrophysiological responses in Wistar albino rats. J Taibah Univ Sci. 49. US Food and Drug Administration. Guidance for Industry: Estimating the 2016;10:505–512. Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult 76. Abu-Taweel GM, Zyadah M, Ajarem JS, et al. Cognitive and biochemical effects Healthy Volunteers. Rockville, MD: US Food and Drug Administration, Center for of and aspartame, administered individually and in Drug Evaluation and Research; https://www.fda.gov/downloads/drugs/guidan combination in male albino mice. Neurotoxicol Teratol. 2014;42:60–67. ces/ucm078932.pdf. Published July 2005. Accessed July 24, 2017. 77. Kim J-Y, Seo J, Cho K-H. Aspartame-fed zebrafish exhibit acute deaths with swim- 50. Reagan-Shaw S, Nihal M, Ahmad N. Dose translation from animal to human stud- ming defects and -fed zebrafish have elevation of cholesteryl ester ies revisited. FASEB J. 2008;22:659–661. transfer protein activity in hypercholesterolemia. Food Chem Toxicol. 51. Fernstrom JD. Oral aspartame and plasma phenylalanine: pharmacokinetic differ- 2011;49:2899–2905. ence between rodents and man, and relevance to CNS effects of phenylalanine. 78. Christian B, McConnaughey K, Bethea E, et al. Chronic aspartame affects T-maze J Neural Transm. 1989;75:159–164. performance, brain cholinergic receptors and Naþ,Kþ-ATPase in rats. Pharmacol 52. Wurtman RJ, Maher TJ. Effects of oral aspartame on plasma phenylalanine in Biochem Behav. 2004;78:121–127. humans and experimental rodents. J Neural Transm. 1987;70:169–173. 79. Abd El-Samad A. Light and electron microscopic study on the effect of aspar- 53. Kotsonis FN, Hjelle JJ. The safety assessment of aspartame: scientific and regula- tame on the cerebellar cortex of male albino rat. Egypt J Histol. tory considerations. In: Tschanz C, Butchko HH, Stargel WW, Kotsonis FN, eds. 2010;33:419–430. The Clinical Evaluation of a : Assessment of Aspartame. Boca 80. Ashok I, Sheeladevi R. Oxidant stress evoked damage in rat hepatocyte leading Raton, FL: CRC Press; 1996:23–41. to triggered nitric oxide synthase (NOS) levels on long term consumption of as- 54. Reynolds W, Stegink LD, Filer L, et al. Aspartame administration to the infant partame. J Food Drug Anal. 2015;23:679–691. monkey: hypothalamic morphology and plasma amino acid levels. Anat Rec 81. Kumar Choudhary A, Selvaraj S, Sheela Devi R. Aspartame induce modification in (Hoboken). 1980;198:73–85. membrane bound and antioxidant enzymes in liver and kidney of Wistar albino 55. Tsakiris S, Giannoulia-Karantana A, Simintzi I, et al. The effect of aspartame rats. Curr Nutr Food Sci. 2014;10:275–287. metabolites on human erythrocyte membrane acetylcholinesterase activity. 82. Iman MM. Effect of aspartame on some oxidative stress parameters in liver and Pharmacol Res. 2006;53:1–5. kidney of rats. Afr J Pharm Pharmacol. 2011;5:678–682. 56. Simintzi I, Schulpis KH, Angelogianni P, et al. The effect of aspartame on acetyl- 83. Martins MRI, Azoubel R. Effects of aspartame on fetal kidney: a morphometric cholinesterase activity in hippocampal homogenates of suckling rats. Pharmacol and stereological study. Int J Morphol. 2007;25:689–694. Res. 2007;56:155–159. 84. Choudhary AK, Sundareswaran L, Devi RS. Aspartame induced cardiac oxidative 57. Simintzi I, Schulpis KH, Angelogianni P, et al. The effect of aspartame metabolites stress in Wistar albino rats. Nutr Clin Metab. 2016;30:29–37. on the suckling rat frontal cortex acetylcholinesterase. An in vitro study. Food 85. Gudadhe DR, Talhar SS, Bokariya P, et al. Histo-morphometric demonstration of Chem Toxicol. 2007;45:2397–2401. cardiotoxic effects of aspartame on mice. IOSR J Pharm. 2013;3:30–33. 58. Ashok I, Sheeladevi R, Wankhar D. Long term effect of aspartame (artificial 86. Choudhary AK, Devi RS. Longer period of oral administration of aspartame on cy- sweetener) on membrane homeostatic imbalance and histopathology in the rat tokine response in Wistar albino rats. Endocrinol Nutr. 2015;62:114–122. brain. Free Radicals and Antioxidants. 2013;3:S42–S49. 87. Choudhary AK, Rathinasamy SD. Aspartame induces alteration in electrolytes ho- 59. Ashok I, Sheeladevi R, Wankhar D. Effect of long-term aspartame (artificial sweet- meostasis of immune organs in wistar albino rats. Biomed Prevent Nutr. ener) on anxiety, locomotor activity and emotionality behavior in Wistar albino 2014;4:181–187. rats. Biomedicine and Preventive Nutrition. 2014;4:39–43. 88. Okasha EF. Effect of long term-administration of aspartame on the ultrastructure 60. Iyyaswamy A, Rathinasamy S. Effect of chronic exposure to aspartame on oxida- of sciatic nerve. J Microsc Ultrastruct. 2016;4:175–183. tive stress in the brain of albino rats. J Biosci. 2012;37:679–688. 89. Palmn€as MS, Cowan TE, Bomhof MR, et al. Low-dose aspartame consumption dif- 61. Villareal LMA, Cruz RAM, Ples MB, et al. Neurotropic effects of aspartame, stevia ferentially affects gut microbiota-host metabolic interactions in the diet-induced and on memory retention and on the histology of the hippocampus of obese rat. PLoS One. 2014;9:e109841. doi:10.1371/journal.pone.0109841 the ICR mice (Mus musculus). Asian Pac J Trop Biomed. 2016;6:114–118. 90. Suez J, Korem T, Zeevi D, et al. Artificial sweeteners induce glucose intolerance 62. Bergstrom BP, Cummings DR, Skaggs TA. Aspartame decreases evoked extracel- by altering the gut microbiota. Nature. 2014;514:181–186. lular dopamine levels in the rat brain: an in vivo voltammetry study. 91. Mohanty J, Nagababu E, Rifkind JM. Red blood cell oxidative stress impairs oxy- Neuropharmacology. 2007;53:967–974. gen delivery and induces red blood cell aging. Front Physiol. 2014;5:84. 63. Vences-Mejıa A, Labra-Ruız N, Hernandez-Martınez N, et al. The effect of aspar- doi:10.3389/fphys.2014.00084 tame on rat brain xenobiotic-metabolizing enzymes. Hum Exp Toxicol. 92. Valko M, Leibfritz D, Moncol J, et al. Free radicals and antioxidants in normal 2006;25:453–459. physiological functions and human disease. Int J Biochem Cell Biol. 64. Abhilash M, Sauganth Paul M, Varghese MV, et al. Long-term consumption of as- 2007;39:44–84. partame and brain antioxidant defense status. Drug Chem Toxicol. 93. Knight JA. Review: free radicals, antioxidants, and the immune system. Ann Clin 2013;36:135–140. Lab Sci. 2000;30:145–158. 65. Abdel-Salam O, Salem N, El-Shamarka M, et al. Studies on the effects of aspar- 94. Cemerski S, van Meerwijk JP, Romagnoli P. Oxidative-stress-induced tame on memory and oxidative stress in brain of mice. Eur Rev Med Pharmacol T lymphocyte hyporesponsiveness is caused by structural modification rather Sci. 2012;16:2092–2101. than proteasomal degradation of crucial TCR signaling molecules. Eur J 66. Abdel-Salam OM, Salem NA, Hussein JS. Effect of aspartame on oxidative stress Immunol. 2003;33:2178–2185. and monoamine levels in lipopolysaccharide-treated mice. 95. Redondo PC, Jardin I, Hernandez-Cruz JM, et al. Hydrogen peroxide and peroxy- Neurotox Res. 2012;21:245–255. nitrite enhance Ca2þ mobilization and aggregation in platelets from type 2 dia- 67. Park CH, Choi SH, Piao Y, et al. Glutamate and aspartate impair memory reten- betic patients. Biochem Biophys Res Commun. 2005;333:794–802. tion and damage hypothalamic neurons in adult mice. Toxicol Lett. 96. Pacher P, Szabo C. Role of the peroxynitrite-poly (ADP-ribose) polymerase path- 2000;115:117–125. way in human disease. Am J Pathol. 2008;173:2–13.

Nutrition ReviewsVR Vol. 75(9):718–730 729 97. Sonnen JA, Larson EB, Gray SL, et al. Free radical damage to cerebral cortex in 115. Villareal LMA, Cruz RAM, Ples MB, et al. Neurotropic effects of aspartame, stevia Alzheimer’s disease, microvascular brain injury, and . Ann Neurol. and sucralose on memory retention and on the histology of the hippocampus of 2009;65:226–229. the ICR mice (Mus musculus). Asian Pac J Trop Biomed. 2016;6:114–118. 98. Haorah J, Ramirez SH, Schall K, et al. Oxidative stress activates protein tyrosine ki- 116. El-Samad AA. Light and electron microscopic study on the effect of aspartame nase and matrix metalloproteinases leading to blood–brain barrier dysfunction. on the cerebellar cortex of male albino rat. Egypt J Histol. 2010;33:419–430. J Neurochem. 2007;101:566–576. 117. Gu Y, Dee C, Shen J. Interaction of free radicals, matrix metalloproteinases and 99. Avshalumov MV, Chen BT, Rice ME. Mechanisms underlying H2O2-mediated inhi- caveolin-1 impacts blood-brain barrier permeability. Front Biosci (Schol Ed). bition of synaptic transmission in rat hippocampal slices. Brain Res. 2010;3:1216–1231. 2000;882:86–94. 118. Gu Y, Zheng G, Xu M, et al. Caveolin-1 regulates nitric oxide-mediated matrix 100. Amor S, Puentes F, Baker D, et al. Inflammation in neurodegenerative diseases. metalloproteinases activity and blood–brain barrier permeability in focal cerebral . 2010;129:154–169. ischemia and . J Neurochem. 2012;120:147–156. 101. Welsh P, Lowe GD, Chalmers J, et al. Associations of proinflammatory cytokines 119. Muriel P, Gordillo KR. Role of oxidative stress in liver health and disease. Oxid with the risk of recurrent . Stroke. 2008;39:2226–2230. Med Cell Longev. 2016;2016:9037051. doi:10.1155/2016/9037051 102. Anaya-Prado R, Toledo-Pereyra LH, Lentsch AB, et al. Ischemia/reperfusion injury. 120. Contreras-Zentella ML, Hernandez-Munoz~ R. Is liver enzyme release really associ- J Surg Res. 2002;105:248–258. ated with cell necrosis induced by oxidant stress? Oxid Med Cell Longev. 103. Montalvo-Jave EE, Escalante-Tattersfield T, Ortega-Salgado JA, et al. Factors in 2016;2016: 3529149. doi:10.1155/2016/3529149 the pathophysiology of the liver ischemia-reperfusion injury. J Surg Res. 121. Muriel P. Role of free radicals in liver diseases. Hepatol Int. 2009;3:526–536. 122. Kawaratani H, Tsujimoto T, Douhara A, et al. The effect of inflammatory cytokines 2008;147:153–159. in alcoholic liver disease. Mediators Inflamm. 2013;2013:495156. doi:10.1155/ 104. Selzner N, Rudiger H, Graf R, et al. Protective strategies against ischemic injury of 2013/495156 the liver. Gastroenterology. 2003;125:917–936. 123. Kim J, Seok YM, Jung K-J, et al. Reactive oxygen species/oxidative stress contrib- 105. Wheeler MD. Endotoxin and Kupffer cell activation in alcoholic liver disease. utes to progression of kidney fibrosis following transient ischemic injury in mice. Alcohol Res Health. 2003;27:300–306. Am J Physiol Renal Physiol. 2009;297:F461–F470. 106. Koeppen BM. The kidney and acid-base regulation. Adv Physiol Educ. 124. Vianna HR, Soares CMBM, Tavares MS, et al. Inflammation in chronic kidney dis- 2009;33:275–281. ease: the role of cytokines. J Bras Nefrol. 2011;33:351–364. 107. Sureshbabu A, Ryter SW, Choi ME. Oxidative stress and autophagy: crucial modu- 125. Fowler SP. Low-calorie sweetener use and energy balance: results from experi- lators of kidney injury. Redox Biol. 2015;4:208–214. mental studies in animals, and large-scale prospective studies in humans. 108. Barodka VM, Nagababu E, Mohanty JG, et al. New insights provided by a com- Physiol Behav. 2016;164(pt B):517–523. parison of impaired deformability with erythrocyte oxidative stress for sickle cell 126. Shen MJ, Zipes DP. Role of the autonomic nervous system in modulating cardiac disease. Blood Cells Mol Dis. 2014;52:230–235. arrhythmias. Circul Res. 2014;114:1004–1021. 109. Huertas A, Das SR, Emin M, et al. Erythrocytes induce proinflammatory endothe- 127. von Borell E, Langbein J, Despre´s G, et al. Heart rate variability as a measure of lial activation in . Am J Respir Cell Mol Biol. 2013;48:78–86. autonomic regulation of cardiac activity for assessing stress and welfare in farm 110. Cemerski S, Cantagrel A, van Meerwijk JP, et al. Reactive oxygen species differen- animals—a review. Physiol Behav. 2007;92:293–316. tially affect T cell receptor-signaling pathways. J Biol Chem. 128. Carney RM, Freedland KE, Stein PK, et al. Heart rate variability and markers of in- 2002;277:19585–19593. flammation and coagulation in depressed patients with coronary heart disease. J 111. Thore´n FB, Betten A˚, Romero AI, et al. Cutting edge: antioxidative properties of Psychosom Res. 2007;62:463–467. myeloid dendritic cells: protection of T cells and NK cells from oxygen radical- 129. Victor VM, De La Fuente M. Immune cells redox state from mice with endotoxin- induced inactivation and apoptosis. J Immunol. 2007;179:21–25. induced oxidative stress. Involvement of NF-jB. Free Radic Res. 2003;37:19–27. 112. FitzGerald GA. Translational therapeutics at the platelet vascular interface: intro- 130. Paiva CN, Bozza MT. Are reactive oxygen species always detrimental to patho- duction. Arterioscler Thromb Vasc Biol. 2008;28:S3–S4. gens? Antioxid Redox Signal. 2014;20:1000–1037. 113. Mariani E, Polidori M, Cherubini A, et al. Oxidative stress in brain aging, neurode- 131. Pepino MY, Bourne C. Non-nutritive sweeteners, energy balance, and glucose generative and vascular diseases: an overview. J Chromatogr B Analyt Technol homeostasis. Curr Opin Clin Nutr Metab Care. 2011;14:391–395. Biomed Life Sci. 2005;827:65–75. 132. Prashant G, Patil RB, Nagaraj T, et al. The antimicrobial activity of the three com- 114. Euston DR, Gruber AJ, McNaughton BL. The role of medial prefrontal cortex in mercially available intense sweeteners against common periodontal pathogens: memory and decision making. Neuron. 2012;76:1057–1070. an in vitro study. J Contemp Dent Pract. 2012;13:749–752.

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