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1821Biochemical responses and mitochondrial mediated activation of apoptosis on long-term effect of aspartame in rat brain, Iyaswamy Ashok, Rathinasamy Sheeladevi, U of Madras 2014.04.29 free full text: Rich Murray 2014.05.24

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  • Rich Murray
    May 24, 2014
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      Biochemical responses and mitochondrial mediated activation of apoptosis on long-term effect of aspartame in rat brain, Iyaswamy Ashok, Rathinasamy Sheeladevi, U of Madras 2014.04.29 free full text: Rich Murray 2014.05.24

      aspartame harm in rat brain from 75 mg/kg gives human ADI 0.75 mg/kg, 53 times less than EU ADI 40 mg/kg, Ashok Iyyaswamy, SheelaDevi Rathinasamy, U. Madras 2012.08.03 free full text -- main methanol toxin is formaldehyde, not formate: Rich Murray 2013.06.01

      [ See also:

      methanol toxicity, not by formate, but by formaldehyde made inside human cells by ADH1 enzyme -- brief by Woodrow C. Monte, with lengthy references: Rich Murray 2013.05.24

      Rathinasamy Sheeladevi  <drsheeladeviibms@...>

      free full text

      Redox Biology
      Available online 29 April 2014

      In Press, Accepted Manuscript — Note to users

      Cover image
      Open Access
      Research Paper

      Biochemical responses and mitochondrial mediated activation of apoptosis on long-term effect of aspartame in rat brain.

      Iyaswamy Ashok,
      Rathinasamy Sheeladevi, Corresponding author
      contact information,
      E-mail the corresponding author

      Aspartame administration alters the functional activity in the brain by elevating the antioxidant levels.
      Chronic aspartame consumption altered the neuronal function and neurodegeneration in brain.
      Observed changes may be due to the methanol or its metabolite.
      Long-term FDA approved daily acceptable intake (40 mg/kg bwt) aspartame administration distorted the brain function and generated apoptosis in brain regions.

      Aspartame, an artificial sweetener is very widely used in many foods and beverages. But there are controversies about its metabolite which is marked for its toxicity. 
      Hence it is believed to be unsafe for human use. 
      Previous studies have reported on methanol exposure with involvements of free radicals on excitotoxicity of neuronal apoptosis.
      Hence, this present study is proposed to investigate whether chronic aspartame (FDA approved Daily Acceptable Intake (ADI),40 mg/kg bwt) administration could release methanol, whether it can induce changes in brain oxidative stress status and gene and protein expression of anti-apoptotic Bcl-2 and pro-apoptotic Bax and caspase-3 in the rat brain region. 
      To mimic the human methanol metabolism, methotrexate (MTX)-treated Wistar strain male albino rats were used and after the oral administration of aspartame, the effects were studied along with controls and MTX-treated controls. 
      Aspartame exposure resulted with a significant increase in the enzymatic activity in protein carbonyl, Lipid peroxidation levels, Superoxide dismutase, Glutathione-S-Transferase, Glutathione peroxidase and Catalase activity in (Aspartame MTX)-treated animals and with a significant decrease in reduced Glutathione, Glutathione reductase and protein thiol, pointing out the generation of free radicals. 
      The gene and protein expression of pro apoptotic marker Bax showed a marked increase whereas the anti-apoptotic marker Bcl-2 decreased markedly indicating the aspartame is harmful at cellular level. 
      It is clear that long term aspartame exposure could alter the brain antioxidant status, and can induce apoptotic changes in brain.

      Free radical; Oxidative stress; Antioxidant; Apoptosis; Aspartame; Mitochondria

      1. Introduction

      Aspartame (L-aspartyl-L-phenylalanine methyl ester) is a low calorie artificial sweetener consumed by 200 million people worldwide. 
      In 1965 aspartame was discovered by James Schlatter and was approved for use by the FDA in the 1970s. 
      This sweetener is added to many soft beverages, cakes etc., and its usage is increasing in health conscious societies as used in the weight reduction regime [1]. Upon ingestion approximately 50% of the aspartame molecule is phenylalanine, 40% is aspartic acid and 10% is methanol [2]. 

      Based on the literature study Kruse [3] suggested that among the metabolites, methanol is a toxicant that causes systemic toxicity. 
      Parthasarathy et al. [4], reported that methanol is primarily metabolized to formaldehyde and then to formate, accompanied by the formation of superoxide anion and hydrogen peroxide. 
      There are a few reports on aspartame consumption on various neurological effects that include headache, insomnia and seizures [5], alterations in regional concentrations of catecholamine [6] which is accompanied with behavioral disturbances [7]. 
      The accumulation of formate rather than methanol is itself considered to cause methanol toxicity [8]. 
      In addition to that, inhibition of cytochrome oxidase by formate also leads to the generation of superoxide, peroxyl and hydroxyl radicals [9]. 

      Methanol intoxication is associated with mitochondrial damage and increased microsomal proliferation, resulting in increased production of oxygen radicals [10,11]. 
      Oxidative stress is defined as an imbalance between the elevated level of reactive oxygen species (ROS) and / or impaired function of the antioxidant defense system. Aspartame can affect the brain and their by the behavior [12]. 
      Free radical overproduction directly causes death of immature cultured cortical neurons [13] and directly induces DNA damage [14].

      Of all the organs in the body, the CNS takes more than its share of oxidative abuse.
      This is associated with the abundance of redox active transition metal ions, and the relative death of antioxidant defense system [15]. 
      Chandra et al. [16], reported that Reactive oxygen species (ROS) can trigger apoptosis. Beside high oxygen consumption and presence of high levels of polyunsaturated fatty acids (PUFA), the brain can be the target for free radicals [17] makes them even more venerable to oxidative stress. 
      There is accumulating evidence of a direct involvement of the cellular redox status in the activation and the functioning of the apoptosis machinery [18].
      Scaiano et al. [19], has reported that free radicals were responsible for induction of cellular damage that leads to chromosomal aberrations. 
      Apoptosis, a form of programmed cell death, plays an important role in embryogenesis and in the normal development and maintenance of many adult tissues [20,21]. 
      Apoptosis is tightly regulated by the expression or activation of several genes and proteins [22]. 
      The initiation and execution of apoptosis depend on activation of the receptor and/or mitochondrial-dependent death pathways [[23], [24] and [25]].
       The Bax (Bcl-2 associated X protein) gene was the first identified pro-apoptotic member of the Bcl-2 protein family (B-cell lymphoma-2) [26]. 
      In the nervous system, Bcl-2 protects against various stimuli that induce apoptotic neuronal death [27,28]. 
      The mitochondrial Bcl2 gene family of proteins has been demonstrated to be important for regulating apoptosis induced by a variety of stimuli [29,26]. 
      Caspases, (cysteine-aspartic proteases or cysteine-dependent aspartate-directed proteases) are a family of cysteine proteases that play essential roles in apoptosis (programmed cell death). 
      Caspase-3 activation may play a key role in triggering apoptosis in neuronal cells has been also suggested by Stefanis et al. [30].

      Due to the high liver folate content, rodents do not develop metabolic acidosis during methanol poisoning as formate is metabolized quickly.
      Only folate deficient rodents are required to accumulate formate in order to develop acidosis and to study methanol poisoning [31,32]. 
      Hence, in this study, to mimic the human situation, a folate deficiency status was induced by administering methotrexate (MTX) and folate deficient diet.
      Many concerns have been raised about the side effects of aspartame consumption and its safety. 
      Since in our earlier study report (for 75 mg/day aspartame) showed a marked increase in blood methanol level, whether the chronic oral administration of aspartame (40 mg/kg bwt) can also accumulate methanol after metabolism, and further to investigate the antioxidant status in brain and there by any alteration in the apoptotic genes forms the basis of this study.

      5. Discussion

      The present study confirms that daily oral administration of 40 mg/kg of aspartame for 90 days, led to alterations in the antioxidant status by inducing free radicals in the brain. Ashok and Sheeladevi [51] reported chronic exposure to aspartame (75 mg/kg bwt) induced detectable methanol level in blood.
      In this study, the blood methanol was elevated markedly after aspartame ingestion (40 mg/kg bwt) which is the Daily acceptable intake permitted level.
      Ishak et al. [52], reported that the derivatives of such metabolism are sometimes more toxic than the initial substance which is true in the case of aspartame as methanol is released after metabolism.
      According to Jeganathan and Namasivayam [53] methanol is toxic to brain as the increased blood methanol level can lead to severe shifts in brain monoamine levels.
      It is well known that the nervous system is highly susceptible for methanol intoxication. Due to the high liver folate content, rodents do not develop metabolic acidosis during methanol poisoning as formate is metabolized quickly. 
      Only folate deficient rodents are required to accumulate formate in order to develop acidosis and to study methanol poisoning [31,32]. 
      Hence, in this study, to mimic the human situation, a folate deficiency status was induced to study the methanol toxicity upon aspartame administration.

      5.1. Free radical generation by methanol the toxic metabolite

      The free radical increase in the present study may be due to the methanol that has been released during aspartame metabolism, as the pathway leading to formate by catalase system is discontinued by MTX administration.

      Maria et al. [54], provided the conclusive evidence about the generation of methanol-derived free radical metabolites.
      Goodman and Tephly [55] reported that methanol is metabolized via three enzyme systems, namely the alcohol dehydrogenase system, the catalase per oxidative pathway and the microsomal oxidizing systems.
      Among these microsomal oxidizing system is reported to be responsible for free radical generation. 
      In this study as folate deficient animals are used to mimic the human metabolism of methanol the catalase per oxidative pathway is much declined.

      The cells are generally protected with an extensive antioxidant defense system. 
      In this study the aspartame-treated MTX animals increased lipid peroxidation with alteration in the enzymatic and non-enzymatic scavenging system warrant the generation of free radicals.
      The increase in free radicals could not be ignored as cells can be injured or killed when the ROS generation overwhelms the cellular antioxidant capacity [56].
      The increase in the lipid peroxidation could not be neglected as it is an auto catalytic mechanism leading to oxidative destruction of cellular membranes [57].
      Lipid peroxidation is initiated by the abstraction of a hydrogen atom from the side chain of polyunsaturated fatty acids in the membrane [58]. 
      The presence of lipid per oxides in a membrane disrupts its function by altering fluidity and allowing ions such as Ca2+ to leak across the membrane and major contributor to the loss of cell function [59].

      5.2. Free radical scavenging systems

      Antioxidants and free radical scavenging systems exist in the cells to protect it against the damaging effects of free radicals [60]. 
      The present study showed that, the oral administration of aspartame could lead to a significant elevation in SOD, CAT, GPx compared to control group.
      In spite of the increase in SOD activity the elevation of lipid peroxidation specify the increased production of free radicals. 
      Increased SOD activity could naturally accumulate the super oxides, H2O2 and justify the increase in CAT and GPx for their increased activity after aspartame ingestion. Enhanced superoxide dismutase activity catalyzes the conversion of superoxide anions to H2O2 which in turn could stimulate the second line of defense which includes glutathione peroxidase and catalase [61].
      The increased SOD levels were only partially effective in combating the oxidative damage [62].

      In free radicals production, the thiol glutathione (glycyl-glutamic acid-cysteine) is the most important cellular free radical scavenging system in the brain [63]. 
      Gebiki and Gebiki [64] reported that free radicals induce the formation of the protein peroxides.
      Glutathione reductase plays an important role in cellular antioxidant protection by catalyzing the reduction of GSSG to GSH [65]. 
      The reduced glutathione reductase activity observed may be one of the reason for the decrease in GSH level observed in the aspartame treated animals. 
      There was a decrease in reduced GSH level observed in the MTX- aspartame treated group, since methanol metabolism depends upon reduced GSH. 
      GSH is a cofactor needed for methanol detoxification [66]. 
      Protein carbonyl content is actually the most general indicator and far the most commonly used marker of protein oxidation [67].
      In this study, there was an increase in the LPO, protein carbonyl levels and a marked reduction in the protein thiol groups. 
      According to Patsoukis et al. [68], and Nikolaos et al. [69], the decreased protein thiol in brain is due to the oxidative damage and well supporting the present findings.
      Abhilash et al. [70], also reported a similar significant decrease in GSH concentration and glutathione activity in rats brain following aspartame consumption (the dosage used by him was 500 and 100 mg/kg). 
      It is essential to point out that even with the FDA approved dosage of (40 mg/kg) similar alteration in the scavenging system was observed.

      5.3. Free radical and markers of apoptosis

      There is accumulating evidence of a direct involvement of the cellular redox status in the activation and the functioning of the apoptosis machinery [18]. 
      It is well known that neuronal death occurs according to an apoptotic program [71]. There is an increase pro-apoptotic Bax and caspases-3 expression with a marked decrease in the Bcl-2 expression in aspartame with MTX treated group compared to controls. 
      Bcl-2 is a key regulator of apoptosis, promotes cell survival either by inhibiting factors that activate caspases [72] or regulating apoptosis by antagonizing the formation of heterodimers with other Bcl-2 family members. 
      Bcl-2 family proteins can indirectly regulate the activity of caspases in related apoptotic pathways [73].
      Bax, a pro-apoptotic member, on the other hand, binds to the anti-apoptotic Bcl-2 protein and thus acts by antagonizing the function of Bcl-2 to revoke apoptosis.
      The salient activation of apoptotic markers such as Bax and caspases-3 and decreasing the regulator Bcl-2 by aspartame could not be overlooked. 
      Because, induction of Bax is also reported to promote cytochrome c release from the mitochondria that eventually leads to apoptosis [74]. 
      According to Mbazima et al. [75], a simultaneous down-regulation of Bcl-2 protein and an up-regulation of Bax in neuronal cells and altering their ratio are in favor of apoptotic cell death.

      Caspases are closely associated with apoptosis. 
      The caspase-cascade system plays vital role in the induction, transduction and amplification of intracellular apoptotic signals. 
      Caspase-3, a key factor in apoptosis execution, is the active form of caspase-3. 
      A depletion of intracellular GSH has been reported to occur with the onset of apoptosis [76,77].
      Hence the GSH depletion after aspartame administration observed in this study may be an additional factor for the apoptotic changes observed. 
      In this study there was increased activated caspase 3 as well as its protein expression with a decrease in reduced GSH and Bcl-2 in aspartame treated animals when compared to control and MTX treated control.

      The relation between free radicals and disease can be explained by the concept of ‘oxidative stress’ elaborated by Sies [78]. 
      Free radicals can also attack DNA strand to induce breaks and base modifications that can lead to point mutation [79]. 
      Scaiano et al. [80], reported that free radicals were responsible for induction of cellular damage that leads to chromosomal aberrations.
      AlSuhzibani [81], reported that aspartame induce a significant increase of chromosome aberration frequencies in mice compared to control providing a supporting scientific evidence that aspartame is toxic.
      The involvement of free radicals with tumor suppressor genes and proto-oncogenes suggest their role in the development of different human cancers [82,83].
      The report of Soffritti et al. [84], highlighted that aspartame can induce cancer. 
      Our previous study also well documented at 75 mg/kg b wt of aspartame treatment corroborated the free radical generation and behavioral changes [85,86].
      Hence, probably the free radicals accumulated due to altered free radical scavenging enzymatic and non-enzymatic system for the alteration observed in the cellular pro and anti-apoptotic markers in discrete brain regions.
      The impact of aspartame induced changes in brain is well represented in the histology of hippocampal region. 
      The aspartame treated animals showed a neuronal shrinkage of hippocampal layer due to degeneration of pyramidal cells in this study. 
      The abnormal neuronal morphology of pyramidal cell layers of Cornu Ammonis was also associated with the disorganized pyramidal cell layers. 
      The present study substantiates that down regulation of Bcl2 and up regulation of Bax with activation of caspase 3 lead to the apoptotic damage of neuronal cells in the brain regions, thus lending support to our present findings.

      It may be concluded that aspartame exposure causes increased production of free radicals and increased oxidative damage to proteins in brain taken for the study. Increased free radicals and consequent increase in the protein oxidative damages might be playing a significant role in the apoptotic neuronal death leading to the development of neuronal toxicity in long-term aspartame exposure.

      6. Conclusion

      This study provide a scientific evidence to conclude that aspartame is toxic to the body system and particularly in brain it increase the free radicals and triggers the apoptosis. Aspartame consumption in a long-term basis may affect the brain. 
      It may be due to its metabolite methanol.
      Aspartame may act as a chemical stressor as indicated by the corticosteroid level.
      Still more studies are required to understand more about aspartame. 
      Now accumulating data from scientific research must reach the public as aspartame is freely available in pharmacy/ super market and consumed largely by diabetic as well as young people who want to reduce weight.

      Conflicts of interest:
      The authors declare that they have no conflicts of interest concerning this article.

      The author is grateful to the suggestion offered by Dr. NJ Parthasarathy and Co-authors. The author is grateful to the help given by the molecular laboratory, department of genetics and endocrinology.
      The financial assistance provided by the Indian Council of Medical Research (ICMR) for Senior Research Fellow, is gratefully acknowledged.
      I acknowledge University of Madras for providing the infrastructure to conduct the research.[  not in 263 page EFSA 2013.12.10 aspartame review  ]

      Parthasarathy JN, Ramasundaram SK, Sundaramahalingam M,
      Pathinasamy SD, Devi RS (2006).
      Methanol induced oxidative stress in rat lymphoid organs.
      J. Occup. Health, 48: 20-27.

      8 pages free full text [ 2.37 g/kg = 287 times more than 8.25 mg/kg ]
      183.  Parthasarathy N, Kumar R, Manikandan S, Devi R.
      Methanol-Induced Oxidative Stress in Rat Lymphoid Organs.
      J Occup Health 2006;48:20-7.

      the first to dig the mine get to share the gold -- 7 recent similar animal research studies indicating much lower aspartame and methanol ADI levels: Rich Murray 2013.12.23

      more lower aspartame and methanol ADIs from studies by RH Nair, SheelaDevi Rathinasamy, WC Monte, PS Jeganathan, A Namasivayam, Hazleton Labs, Searle Labs: Rich Murray 2013.06.01

      James McDonald to EFSA, outdated aspartame ADI gives methanol 35 times too high for human safety, ten minute talk at April 9 public sharing, Brussels: Rich Murray 2013.04.15

      California OEHHA sets methanol ingestion level 23 mg daily, same as from 1 can aspartame diet soda, 10 cigarettes, 3 tomatoes, or 4 cans green beans: Rich Murray 2013.07.03

      "However, the anticipated exposure to methanol from consumption of aspartame would not be considered an exposure within the meaning of Proposition 65 because aspartame is not listed under Proposition 65."

      [ Rich Murray: Many pregnant women drink one 12-oz can aspartame diet drink daily, with 200 mg aspartame that gives 11% methanol, 22 mg, which is just under the OEHHA limit of 23 mg daily.

      The smoke from 10 cigarettes gives 20 mg methanol, the same as from 1 can aspartame drink, 3 full size fresh tomatoes, or 4 cans of unfresh green beans. ]

      smoke from pack cigarettes gives 40 mg methanol for 20 gr tobacco, 6 tobacco methanol papers, Carl Neuberg 1926-1939, Berlin -- so methanol formaldehyde toxicity paradigm is co-factor in 18 tobacco diseases -- WC Monte gives 23 references: Rich Murray 2013.03.29

      Table 5.2 is the key chart -- ADH1 enzyme at high levels in 20 tissues
      in body and fetus makes methanol into formaldehyde right inside cells,
      initiating over 20 human diseases, with full text references, WC Monte
      paradigm: Rich Murray 2013.03.21

      WC Monte finally got secret FDA memo 37 years after Searle Co. labs
      found birth defects in rabbits from aspartame (methanol, becomes
      formaldehyde via ADH1 enzyme within human cells) and its
      phenylalanine: Rich Murray 2012.06.02

      highly competent, pithy analysis of aspartame cancer study by Eva S.
      Schernhammer at Harvard, William R. Ware, PhD, showing relevance of
      Woodrow C. Monte methanol-formaldehyde toxicity paradigm: Rich Murray

      confirms WC Monte paradigm: ingested methanol becomes toxic
      formaldehyde-induced hydroxymethyl DNA adducts in all tissues in rats,
      sensitive C13 test, Kun Lu, James A Swenberg, UNC Chapel Hill
      2011.12.08 Toxicol Sci: Rich Murray 2013.01.11

      methanol (11% of aspartame), made by body into formaldehyde in many
      vulnerable tissues, causes modern diseases of civilization, summary of
      a century of research, Woodrow C Monte PhD, Medical Hypotheses
      journal: Rich Murray 2009.11.15
      Sunday, November 15, 2009

      "Formaldehyde produced within the cell immediately reacts with water
      to produce formal hydrate,[#27] a strong acid[#114] with twice the
      number of available hydrogen ions as the next methanol metabolite,
      formic acid.

      Formal hydrate produced from methanol by the ADH I sites found in
      the intima, media, and adventitia lining of the circulatory system of the
      heart and brain[#220] would be expected to diffuse into the localized
      tissue, quickly methylating basic molecules such as myelin basic
      protein (MS)[#224] and tau protein (Alzheimer's).[#234]"

      free full text, 5 pages
      Monte WC.
      Methanol: A chemical Trojan horse as the root of the inscrutable U.
      Med Hypotheses 2010;74(3):493-6.

      two studies by Rong-Qiao He teams in China on monkeys and mice show oral methanol leads to specific formaldehyde harm similar to Alzheimers disease, confirming WC Monte paradigm: Rich Murray 2014.05.16

      "As a matter of course, every soul citizen of Earth has a priority to quickly find and positively share evidence for healthy and safe food, drink, environment, and society."

      within the fellowship of service,

      Rich Murray,
      MA Boston University Graduate School 1967 psychology,
      BS MIT 1964 history and physics,
      1039 Emory Street, Imperial Beach, CA 91932