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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 s ecreted 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.

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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 l994