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Tuesday, May 10, 2011

MORE ASPARTAME STUDIES SHOWING DAMAGE (Fetal, Brain, etc.) - Peer Reviewed and Published

Aspartaam Onderzoek In Noorwegen
NEUROPHARMACOLOGY AND NEUROTOXICOLOGY
Volume 6 1995 (PP318-320) Rapid Communications of Oxford Ltd
Effects of aspartame on Ca influx and LDH leakage from nerve cells in culture Ursula Sonnewald, Tomm Muller, Geirmund Unsgard, S.B. Peterson MR-Centre, SINTEF UNIMED, N-7034 Trondheim; University of Trondheim, Dept. of Neurosurgery, University Hospital N-7006 Trondheim; Norwegian Institute of Tecnology, Drpt. of Biotecnology, N- 7034 Trondheim, Norway.
Aspartame (ASM), an artificial sweetener, was shown to dose dependently increase CA influx into and lactate dehydrogenase (LDH) leakage from murine brain cell cultures. Astrocytes were more resistant than neurones to the effects of ASM. In cerebellar granule neurones, a 20% increase in calcium was found after an incubation time of 22 h in the presence of 0.1 mM ASM; at 0.5 mM concentration, calcium influx increased 40% compared with control cultures. At a concentration of 10mM, influx was increased 13-fold after 5 h. Morphological appearance as judged by phase contrast microscopy was first visibly affected after exposure to 1mM ASM for 22 h. Citrate, another food additive, was included in the study to demonstrate that cerebellar granule neurones could tolerate 10mM additions to the medium and citrate did not cause Ca influx or morphological changes in neurones after 22 h. LDH leakage, a sign of severe cell damage, was observed at 1 mM concentrations of ASM after 22 h. Cerebral astrocytes on the other hand were more resistant and showed morphological changes, increased calcium influx and LDH leakage first at 5 mM concentrations of ASM.
Keywords:
Aspartame, Neurotoxicity, Cerebellar granule neurones, Lactate dehydrogenase leakage (LDH) Calcium influx
INTRODUCTION
Aspartame (L-aspartyl--L-phenylalanine methyl ester, ASM) is a widely used artificial sweetener in soft drinks and low calorie food. There have been reports of adverse neurological effects such as headache (1), insomnia and seizures after ingestion of aspartame, which may be attributed to the alterations in regional concentrations of catecholamines.(2) Brain phenylalanine and tyrosine were increased following ASM ingestion. (3) Studies using radioactively labelled aspartame in comparison with labelled methanol, aspartame and phenylalanine have shown the 30-40% of the total dose of aspartame of the labelled components remains in the body after 8 h; the remainder is primarily secreted through expired air. (4) Analysis of tissue distribution of orally administered isotopically labelled aspartame in the rat showed part of the label remaining in the brain for up to 24 h. (5) From these studies it was not possible to determine whether ASM or its degradation products reached the brain.
Both aspartate (6) and aspartame (7) have been shown to have excitatory activity. Olney et al (8) have shown that systemic administration of glutamatae, an excitatory amino acid, produced brain damage in a number of animal species including primates, and excitotoxic analogues such as aspartame had the same effects. (9)
In order to investigate potential toxicity of aspartame on brain cells, lactate dehydrogenase leakage and (45) Ca influx into astrocytes and neurones were measured after incubation with varying concentrations of aspartame.
Materials and Methods
Plastic tissue culture dishes were purchased form NUNC A/S (Denmark), fetal calf serum from Seralab (Sussex, UK), poly-L-lysine (mol wt. 300 000) and amino acids from Sigma (St. Louis, MO) ; 45Ca was from Amersham. All other chemicals were of the purest grade available from regular commercial sources.
Cortical astrocytes were cultured essentially as described by Hertz et al. (10) Prefrontal cortex was taken from newborn NMRI mice and passed through Nitex nylon sieves (80 um pore size) into a slightly modified Dulbecco's medium (DMEM) containing 20% (v/v) fetal calf serum and plated in NUNC 3 cm culture dishes. Medium was changed twice a week. Cells were used for experiments after 2-3 weeks in culture. Cerebellar granule cells were prepared from 7-day-old mice; (11) they have been shown to possess NMDA receptors (12) and are useful in the study of neurotoxicity. (12) Tissue samples of cerebella were exposed to mild trypsinization followed by trituration in a DNAse solution containing a soyabean trypsin inhibitor. Cells were suspended (2-3 x 106 cells ml-1) in a slightly modified DMEM with 10% (v/v fetal calf serum. Cytosine arabinoside (20 uM) was added after 48 h to prevent astrocyte proliferation. Cells were used after 7 days in culture. Prior to experiments, the incubation medium was removed and substituted with Hanks balanced salt solution without MG2+ (HBBS) containing 1.5 uCi ml-1 (45)Ca. The experiments were terminated by the removal of the incubation medium. The cells were washed five times with ice-cold phosphate-buffered saline containing 25 mM MgCl2 to displace (45) Ca bound extra-cellularly. The cells were lysed in 0.5 M HCL and the (45) Ca content was determined by liquid scintillation spectrometry. When appropriate, cell integrity in the cultures was assessed by determination of leakage of lactate dehydrogenase (LDH< EC 1.1.27) from cells into the medium, using a diagnostic kit supplied by Sigma Chemical (catalogue no. DG 1340-K). LDH was measured in cell extracts and medium and expressed as percentage of total LDH ((14)
Results and Discussion
Aspartame has been shown to dose-dependently inhibit L-(3H) glutamate binding to the N-methyl-D-aspartame (NMDA) receptor in a synaptosomal preparation from rat brain. (7) The NMDA receptor is an ionotropic glutamate receptor mediating calcium influx into neurones. Aspartate, a constituent of ASM, is a potent NMDA agonist and has been shown to induce widespread late neuronal degeneration. (14) Delayed cell death mediated by the NMDA receptor depended on the presence of extracellular calcuium. (15-17) Thus the present study was undertaken to evaluate the effect of ASM on primary nerve cell cultures in terms of calcium influx. Furthermore measurement of LDH activity released to the extracellular media has been found to be a quantitative method for determining neuronal cell injury. (18) Table 1 shows that ASM dose-and time-dependently increase calcium influx into and LDH leakage from cerebellar granule neurones. No effect was detected at 0.1 mM, but at 0.5 mM ASM LDH leakage was increased slightly and at a concentration of 5 mM LDH leakage was increased by a factor of 2.5 after 22 h (Table 1). After this time cells had detached from the culture dishes and intracellular (45)Ca could not be determined. At 10 mM, calcium influx was increased 13-fold after a 5 h incubation (Table 2). Citrate, another food additive, was included in the study to demonstrate that cerebellar granule neurones could tolerate addition of organic substances at 10 mM concentration to the medium and citrate did not cause (45) Ca influx or morphological changes in neurones; however, deleterious effects on astrocytes were seen. The above findings further confirm the hypothesis of Pan-How et al (7) that the neurotoxicity produced by ASM is mediated by a calcium coupled receptor. In the case of cerebellar granule neurones it is likely to be an NMDA receptor-mediated effect. The excitotoxin responsible for this effect could either be free aspartate (an NMDA receptor agonist) derived from proteolytic cleavage of ASM or ASM directly. Astrocytes on the other hand are not believed to have NMDA receptors and the observed calcium influx at 5 mM ASM (Table 1) must therefore be mediated through a different mechanism. LDH leakage, a sign of cell damage,was also observed in astrocytes (Table 1). Thus it has been shown that ASM has adverse effects both on glia and neurones in culture.
Clearly the concentrations used in these studies are not likely to be physiological, but subpopulations of neurones might be affected by lower doses, and long term exposure to low concentrations might produce cumulative irreversible damage. Based on the results presented here, we cannot draw any conclusions for the in vivo situation, there is the need for additional in vitro and in vivo studies, to evaluate the safety of this food additive that is consumed in increasing amounts by adults and children.
References

1. Johns Dr. Migraine provoked by aspartame. N Engl J Med 315, 456 (1986)
2. Coulomb, RA and Sharma RS. Neurobiochemical alterations induced by the artificial sweetener aspartame. Toxicol Parmacol 83d, 79-85 (1986)
3. Fernstrom JD, Fernstrom MH and Gillis MA. Acute effects of aspartame on large neutral amino acid and monoamines in rat brain. Life Sci 32, 1651-1658 (1983)
4. Opperman JA. Aspartame metabolism in animals. In Stegink LD and Filer Jr. eds. Aspartame Physiology and Biochemnistry. New York: Marcel Dekker, 1984: 161-200.
5. Matsuzawa Y and O'Hara Y. Tissue distribution of orally administered isotopically labelled aspartame in the rat. In. Stegink LD and Filer Jr. eds. Aspartame Physiology and Biochemistry. New York: Marcel Dekker, 1984; 161-200
6. Watkins JC. Excitatory amino acid and central synaptic transmission. Trends Pharmacol 5 373-376 (1984)
7. Pan-Hou H, Ohe Y, Sumi M et al. Effect of aspartame on NMDA sensitive L-(3H)glutamate binding sites in rat brain synaptic membranes. Brain Res 520, 351-353 (1990)

8. Olney Jw. Sharpe LG and Feigin Rd. Glutamate-induced brain damage in infant primates. J Neuropathol Exp eurol 31, 464-488 (1972)
9. Olney JW, Sharpe LG and Feigin RD. Glutamate-induced brain damage in infant primates. J Neuropathol Exp Neurol 31, 464-488 (1972)
10. Hertz l, Juurlink BHG, Hertz E et al. Preparation of primary cultures of mouse (rat) astrocytes. IN: Shahar A, De Vellis J, Vernadakis A, Haber B, eds. A dissection and Tissue Culture Manual of the Nervous System New York: Liss, 1989:105-108
11. Schousboe A, Meier E, Drejer J et al. Preparation of primary cultures of mouse (rat) cerebellar granule cells. In Shahar A, De Vellis J, Vernadakis A. Haber B, eds. A Dissection and Tissue Culture Manual of the Nervous System. New York: Liss, 1989: 183-186
12. Lysko PG, Cox JA, Vignano MA et al. Excitatory amino acid neurotoxicity at the N-methyl-E-aspartame receptor in cultured neurones; pharmacological characterization, Brain Res 499, 258-266 (1989)
13. Frandsen AA and Schousbor A. Time and concentration dependency of the toxicity of excitatory amino acids on cerebral neurones in primary culture. Neurochem Int 10, 583-591 (1987)
14. Choi DW. Non-NMDA receptor-mediated neuronal injury in Alzheimer's disease? Neurobial Aging 10, 605-606 (1989)
15. Hartly DM, Kurth MC , Bjerkness L et al. Glutamate receptor-induced (45) Ca2+ accumulation in cortical cell culture correlates with subsequentneuronal accumulation in cortical cell culture correlates with subsequent neuronal degeneration. J Neursci 13 1993-2000 (1993)
16. Sijesjo BK and Bengtsson F. Calcium fluxes, calcium antagonists, and calcium-related pathology in brain ischemia, hypoglycemia, and spreading depression: A unifying hypothesis. J Cereb Blood Flow Metab 9, 127-140 (1989)
17. Eimerl S and Schramm. The quantity of calcium that appears to induce neuronal death. J Neurochem 62 1223-1226 (1994)
18. Koh JY and Choi DW. Quantitative determination of glutamate mediated cortical neuronal injury in cell culture by lactate dehydrogenase efflux assay. J Neurosci Methods 20, 83-90 (1987)
Acknowledgements: This research was supported by the Research Council of Norway. The use of the animal facilities at the University Hospital in Trondheim are gratefully acknowledged.
Received 26 October l994; accepted 25 Nov l9

Second Norway Study:
Aspartame Brain Damage In Mice
See the original athttp://www.aspartaam.nl/artikelen_eng/nordagbl.htm Hetle & Eltervaag: 2001 thesis abstract aspartame brain damage in mice: Sommewald 1995 study.
For thesis in Norwegian, mailed by regular mail, contact: Anne Varnes anne.varnes@medisin.ntnu.no
"Cola light, one calorie" men hva med jhernen?
Hovedfagoppgave hosten 2001 Utfort av Arnstein
Eltervaag og Elisabeth Hetle Det medisinske
fakultet Institutt for kliniske nevrofag Trondheim Norway 10.desember 2001
The 48-page thesis has 35 references, and includes an English abstract. Faculty and helpers listed in the Forword are: Ursula Sonnewald (with 134 items in PubMed since 1988, showing a distinguished research career in biochemical studies of neurotoxins-- one of her studies on aspartame, published 1995 with three partners, Tomm Muller, Geirmund Unsgard, and S.B. Peterson, is given in full at the end of this post, with 18 references, and obviously presents much the same laboratory technique as applied in 2001 in the thesis.), Hong Qu (female qu.hong@phys.ntnu.no), and Bente Urfjell. Obviously, this team has the experience, facilities, funding, faculty support, and motivation to study the biochemistry of aspartame toxicity in detail.
ABSTRACT
Introduction: Aspartame (ASM) is a product that was originally made for diabetics, but today ASM is widely used by healthy people as an artificial sweetener in many food products.
Purpose: The main goal with this research was to see whether ASM was harmful to brain cells (cerebellar granule cells). We wanted to check if the damage to the neurons is connected to the N-methyl-D-aspartate (NMDA)-receptors on these cells.
PROCEDURE
Brain cells from 7 day old mice were used. They were cultured in 24 Petri well dishes, and different quantities of ASM were added. After 7 days, the cultures were analysed by two different tests: Lactate dehydrogenases (LDH) test, which gives a picture of cell death (LDH leakage to the medium in which the cells were cultured). 3-[4,5- dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromid (MTT) test, which can be used to analyse mitochondrial activity in living cells. To test whether the NMDA-receptor was involved in the damage done by ASM, the receptor was blocked by (?)-2-amino-5 phosphonopentanocid (AP5).
RESULTS
Our results showed damage/cell death from an added quantity of 0.06 mg/ml ASM each day for 4 days. As a comparison there is 0.24 mg/ml ASM in Cola Light. MTT- and LDH-tests showed damage to the neurons at an added quantity of 1.5 and 3.00 mg/ml ASM after 22 hours of incubation. The results also show that ASM is in part acting through the NMDA- receptor because AP5 reduced or blocked the damage to the granule cells.
CONCLUSION
In light of these results, our conclusion is that in order to be on the safe side, it should be warned against use of ASM as a food additive, maybe especially in products consumed by children, because NMDA-receptors and the synapses involved also are connected to learning.

http://www.dagbladet.no/print/?/dinside/2001/12/17/301529.html
[A major newspaper in Norway]

FARLIG FOR HJERNEN?
Medisinstudent Elisabeth Hetle (32) har sluttet ? drikke lettbrus, mens medstudent Arnstein Eltervaag (40) aldri har drukket lettbrus.
I edited this into a fairly accurate English version:
DANGER FOR BRAIN?
Medical student Elisabeth Hetle (32) has stopped using aspartame diet sodas, while fellow student Arnstein Eltervaag (40) has never used them.
You can also read this article at: [article on newspage]
http://www.dagbladet.no/dinside/2001/12/17/301529.html
Dagbladet 2001:
hetle@stud.ntnu.no eltervaa@stud.ntnu.no