Content of the neurotoxins cycasin and BMAA in cycad flour prepared by Guam Chamorros.
Environmental Chemicals

Author: Giulia Arrigoni
Date: 03/09/2013


Cycas circinalis is a plant from cycadacee family and cycas gender which grows in South India.
Its seeds are a fundamental resource of food in famine period for Guam Chamorros because of their high nutritional value. But they also contain active compounds, including cycasin (methylazoxymethanol-beta-D-glucoside) and BMAA (beta-methylamino-L-alanine), which may produce neurotoxic effects. Therefore exposure to cycad seeds kernel is an etiologic factor for the western Pacific amyotrophic lateral sclerosis (ALS) and parkinsonism-dementia complex (PDC), also called Guam Dementia which leads to a progressive dementia associated with a extrapiramidal motor disease or with a sudden motoneuronal syndrome similar to ASL. Return of the cycad hypothesis: does the amyotrophic lateral sclerosis/parkinsonism dementia complex (ALS/PDC) of Guam have new implications for global health? Neuropathology and Applied Neurobiology 2005, 2007

The hypothesis of BMAA as etiologic factor wasn’t agreed at first because low dose of toxin (BMAA for example) in washed Cycas flour wasn’t considered enough to cause neurodegenerative disease. This Hypothesis has been reconsidered later, when BMAA biomagnification in Guam system has been discovered, and found in dead by SLA/PDC Chamorro brain tissues but no in those belonging to Chamorro dead by other disease (no neurodegenerative).
All these studies confirm BMAA as etiologic factor of ASL/PDC explaining why its incidence in Chamorros is 50-100 times higer than in other population: large flying fox (pteropus vampyrus) comsumption, who graze on Cycas fruit, may be the cause of high levels of BMAA exposure which lead to the disease.
While Cycasin, a glicoside component, leads to hepatotoxic and carcinogenic effects but minimal neurological effects and it’s easily removed after flour washing, BMAA is mainly concerned in the disease’s pathogenesis.

Beta-N-Methylamino-L-alanine (BMAA)
Neurotoxin beta-methylamino-L-alanine, also known as α- ammino-β-methylamminopropionic acid (MeDAP) and S(+)-m-methyl-α,β-diaminopropionic acid, is a neurotoxic non-essential amino acid produced by all groups of cyanobacteria in the various environments and eco system of the world.
BMAA is a non-essential and non-lipophilic amino acid which is accumulated in living tissues in free or bounded to protein form. It could be incorporated into proteins and lead them to denaturation, in brain tissue could promote formation of protein aggregates and act as an endogenous neurotoxic reservoir that releases free BMAA slowly, bringing to initial and recurrent neurological damage for many years (this mechanism explains the latency period occurring in the Chamorros neurological disease as well).
The incorporation into proteins mechanism isn’t known yet, but it could lead to denaturation (production of abnormal or non functional proteins in protein synthesis, or interfering with neuro receptor metabolism or ionic function).

Free form BMAA, similar to its carbamate, binds glutamate and leads to neuronal degeneration (in vitro) with a mechanism regulated by EAA receptor or glutamate transport activation: BMAA directly binds NMDA receptors (and this is stronger when BMAA is carbamated), producing a glutamate-like molecule.
Carbamated BMAA derivative are both AMPA and NMDA agonist. In dissociated mixed spinal cord growing, low concentration BMAA (30 μM) induces moto-neuronal selective lost by activating AMPA/KAINATO. This exact concentration (lower than that in the previous studies) is responsible of neuronal degeneration, strengthening the hypothesis of BMAA as a cause of moto-neuronal lost. BMAA selectively injures motor neurones via AMPA/kainite receptor activation. Exp Neurol, 2006.
BMAA is a neuro exciting molecule for glutamate iono-trophic receptor (NMDA, AMPA, Kainato) in organo-trophic mouse cerebral cortex cultures.
BMAA activates metabo-trophic receptor as well (mGluR), and it’s one of the most chelating greedy (Cu2+, Zn2+, Ni2+).
Several studies made on cortical cells cultures demonstrated BMAA as neurotoxic both in vivo and in vitro, but higher concentrations lead to neuronal death (1-3 mM). These results, valued by Lobner at al . β-N-methylamino-L-alanine enhances neurotoxicity through multiple mechanisms. Neurobiology of Disease 2007 , demonstrated how, starting from a10μM concentration, BMAA strengthens neural damage induced by different insults (genetic predisposition for e.), and leads to neurodegenerative diseases increasing the damage, fastening the onset and extending the effects.
BMAA has a triple damage system:
1) direct activation of NMDA receptor
2) direct activation of mGluR5 (glutamate receptor)
3) oxidative stress
Liu et al. beta-N-methylamino-l-alanine induces oxidative stress and glutamate release through action on system Xc(-)Exp Neurol 2009(-). discovered how BMAA inhibits cystein/glutamate antiport (Xc (-) system, cystein captation mediated, which leads to glutathione depletion and increases oxidative stress: in cycled model BMAA drives glutamate release by Xc(-) system whose toxicity is caused by activation of mGluR5.

Nunn e Ponnusamy β-N-Methylaminoalanine (BMAA): Metabolism and metabolic effects in model systems and in neural and other tissues of the rat in vitro. 2009 in their experiments found that BMMA inhibits synthesis or stimulates degradation of glutamine starting from glutamate, maybe for the carbamate BMAA activation of NMDA receptor, this would mean that BMAA would compromise the glucose use, the ATP composition and the glutamate-glutamine cycle activity. The no charged nature (2-amino deprotonated group) of BMAA at pH 7.4 probably explains the amino acid brain entry via neutral amino acid carrier. Reactivity of BMAA with pyridoxine-phosphate represents a potentially dangerous characteristic that can contribute to the toxicity of such compounds.
Experiments on rat brain slices discovered:
1) reduction in taurine, serine and glycine concentrations (probably due to inhibition of their synthesis by the BMAA)
2) increased concentrations of ammonium and alanine but not of glutamate (due to the BMAA dismutation),
3) production of methylamine by the toxin (at physiological pH values by means of a reaction between the 2-amino group of BMAA, only partially ionized at physiological pH, and pyridoxal-5-phosphate).

Simple models in vitro and in primates such as those of Spencer et al. (1987) support the toxicity of BMAA.
Neurodegeneration of specific neuronal populations, motor and cognitive dysfunction were induced in mice by administration of washed cycad flour. In contrast with previous studies, Karlsson et al. (2009) have shown:
1 )BMAA placental transfer
2) fetal brain of mice pronounced absorption in 14th day of gestation
3) transfer through the BBB (blood brain barrier) to neonatal brain
4) localization in distinct areas of the brain (hippocampus and striatum).

Repeated treatments with the BMAA (200 or 600mg/kg) in rats, in the neonatal period, during maximum peak of brain growth spurt (BGS, 9th-10th day), led acute changes such as: impairment of motor skills and hyperactivity (which could have been caused by stimulation of the glutamate system by the
BMAA). Disturbances (as alterations in motor skills or general activities) induced by BMAA are due to the difficulty of spatial learning. BMAA exposure during BGS period creates long-term consequences for the performance of adult behaviour functions and BMAA exposure through food or contaminated water could be a very important risk factor for human health.
Summarizing the BMAA neurotoxic activities, we can say that it certainly:
1) actives ionotropic receptors (AMPA, kainate, NMDA)
2) actives metabotropic receptors (mGluR) of glutamate
3) induces neuronal degeneration through an excitotoxic mechanism
4) induces the selective loss of motor neurons in mixed cultures of dissociated spinal cord at concentrations of about 30 mM
5) induces oxidative stress in low concentrations
6) can affect neurite growth and levels of neurofilament protein in cultured cells
7) alters protein function through the incorporation into proteins
8) induces a neuronal degeneration by an excess activation of glutamate receptors
9) from concentrations of 10 mM BMAA potentiates the damage induced by other insults, and in vitro studies have demonstrated the toxicity at 10-30 mM concentrations
10) there is always more belief that excitotoxic mechanisms involving glutamate are part of the the pathogenesis of human neurodegenerative diseases, including ALS and AD.

Latency effects

Since latency time between exposure to BMAA and the onset of neurodegeneration it is not known, the analysis of migrants from Guam could give an indication. Chamorro who emigrated to other Pacific islands and further, rarely develops lytico-Bodig and ALS, PD and AD are not higher than those of the surrounding population. Analysis of the risk of developing ALS / PDC by migrants residing in Guam for the first 18 years of their life, it indicates that the disease may develop up to 35 years or more after the arrival in the new location, but the second generation of these immigrants who were born and reside in the new geographical location, does not show a higher risk for neurodegenerative diseases.
Twenty-eight Chamorro immigrants from Guam have developed ALS / PDC after a period of absence that goes from 1 to 34 years: if environmental factors are responsible for the period of latency, for the development of the disease may therefore be necessary more than three decades. Rough estimates of the rates of mortality from ALS for these migrants are at least three times higher than the rates observed for the population of the United States, but more than four times lower than the rates of mortality for ALS in Guam Chamorro non-migrants living there in the twenty years prior to the study. From the epidemiological point of view this means that the exposure to which migrants were subjected occurred in Guam. The Filipinos migrated to Guam reported to have developed progressive neurological disease after their arrival (10 with ALS from 1 to 32 years after their arrival, 2 with parkinsonism-dementia from 13 to 26 years after arrival, and 7 other patients with the classic PD 5 to 24 years after their arrival). Ten children born in Guam from Filipino parents and Chamorro have developed ALS and six have developed PD. The average annual crude ALS mortality among Filipino migrants was estimated to be six times higher than that living in the continental United States, but half the rate observed among the Chamorros living in Guam in the same period. It is likely to be gene-environment interactions that make some people more sensitive to exposure, so further research is needed to clarify all these points unresolved.

Toxic synergy at low concentration
Studies in cortical cells culture have shown how neurotoxic is BMAA both in vivo and in vitro, but very high concentrations of BMAA (1-3 mM) are required to induce neuronal death.
Lobner et al. β-N-methylamino-L-alanine enhances neurotoxicity through multiple mechanisms. 2007 repeated these studies in cortical cells culture and had similar results, in which BMAA alone does not induce any significant toxicity up to a concentration of 1 mM. Then valuing the effects of the concentrations of 10, 50 and 100 uM of BMAA, both alone or in combination with other insults that induce low levels of neuronal death, was discovered that BMAA at these concentrations does not cause toxicity by itself, but starting from a 10 mM concentrations may potentiate the neural damage induced by other insults. In particular, at 100 uM or less, BMAA potentiates N-methyl-D-aspartate (NMDA), iron (Fe), buthionina sulfoximine (BSO), β-amyloid, and ion 1-methyl-4-phenylpyridine (MPP +) toxicity.
The lowest concentration that resulted in an effect, instead, was a 10 uM of BMAA, which has caused the enhancement of the toxicity of β-amyloid and MPP +. This is the first evidence that BMAA at concentrations below the range of mM can increase the death of cortical neurons, and illustrates the potential synergistic effects of toxins environmental neurological disorders.

BMAA metabolism

Rats treated with BMAA release L-α-N-acetyl derivative, which is the only pathway known of BMAA in animals, and it is formed by N-acetylation using acetyl-CoA, the α-N-acetyl derivatived by BMAA, while treating the BMAA with L-amino acid oxidase led to its oxidation.
The study of metabolism of BMAA in a number of rat tissues in vitro during a two-hour cells incubation, by Duncan et al. did not get results; but after seven days they found less than 10% of an injected dose of [14CH3]-BMAA in rats urine and excrements. This suggested a significant metabolism of this neurotoxin during that period.
Mash et al. (2008) found that the triziated methyl group of BMAA molecule is lost quickly in the liver and kidney of rats in vivo, but persists in the brain.
The metabolism of BMAA was recently studied by Nunn and Ponnusamy β-N-Methylaminoalanine (BMAA): Metabolism and metabolic effects in model systems and in neural and other tissues of the rat in vitro. Toxicon 2009 which examined aspects (by observing the appearance of specific metabolites derived from the amino acid) and metabolic effects of BMAA in a number of rat tissues in vitro, confirming that the toxin is a very reactive molecule in enzymatic and non-enzymatic reactions. This study has provided important information on metabolism of this neurotoxin still little known.


Martin (1978) examined the effects of physical movements of amino groups in diamine mono-carboxylic acids. In BMAA the two amino groups are very close, as in 2,3 diamino-propanoic acid: this feature seems to be the reason why the reactions of the two compounds with bicarbonate and with some metal ions are alike.
The 2,3-diamino-propanoic acid (2,3-DAB) and the BMAA therefore share low pKa values of the two amino groups (pKa2 = 6.50): therefore these molecules do not take a net charge at pH 7. For the BMAA the negative charge on the carbonyl group at pH 7 is balanced by a positive charge distributed between groups 2 and 3-amino (β)-methylamino. The unusually low value of pKa of the 2-amino group results in a low overall net charge on the molecule at physiological pH value. The negative charge on the carboxyl group is balanced by a unit of positive charge shared between the 2-amino and 3 (β)-methylamino groups. The free of charge state of the 2-amino group means that it is free to react in conditions in which an amino group, predominantly positive charged (typical of most of the amino acids in the same conditions), would be reactive β-N-Methylaminoalanine (BMAA): Metabolism and metabolic effects in model systems and in neural and other tissues of the rat in vitro. 2009. The not charged state of BMAA at pH 7.4 probably explains the entry of the amino acid in the brain via neutral amino acid transporter. In most tissues, amino acids react only in catalyzed enzymatic reactions in which the positive charge of the amino group becomes deprotonated. This happens because amino groups with negative charge are not chemically very reactive. Furthermore, the fact that in the physiological environment the 2-amino group of BMAA is almost without a charge explains its reactivity with pyridoxal-5-phosphate, and this represents a potentially dangerous state that can contribute to the toxicity of such compounds. The L-alanine does not react with pyridoxal-5-phosphate at physiological values of pH, because in these conditions the 2-amino group has a positive charge.

1. Effects on brain taurine, glycine, serine and ammonium and alanine

Nunn and Ponnusamy β-N-Methylaminoalanine (BMAA): Metabolism and metabolic effects in model systems and in neural and other tissues of the rat in vitro. 2009 , in their experiments on the brain, found a reduction in taurine serine and glycine concentrations, probably due to inhibition of their synthesis by the BMAA. They also found a increased concentration of the three amino acids in the extracellular environment, probably because BMAA stimulates the loss of these compound by trans-membrane exchange, using specific transport systems or general one. Furthermore taurine and BMAA are structurally very similar in both molecular size and charge distribution. Taurine is a very important amino acid in cell volume regulation and it is accumulated from the extracellular fluid by transport system, like the TauT. The great loss of taurine from brain was probably caused by the different exchange of taurine and BMAA, or even by the stimulation of the release of taurine from the brain stem of immature rat by both ionotropic receptors(AMPA and NMDA). Carbamic derivates of BMAA are agonists of both AMPA and NMDA (receptors of the glutamate).
Another result of these experiments was the increased concentrations of ammonium and alanine both inside the cells and into the extracellular fluid, indicating that these compounds were synthesized within the tissue and subsequently lost from it. On the contrary the glutamate concentration is unchanged both in brain tissue and the environment.

2. Effects on glutamine

The glutamine concentration reduction in both brain slices and in incubation liquids indicates that the BMAA inhibits the synthesis and / or stimulates the degradation of glutamine. Most of the glucose used in the rat and in the Human central nervous system in vivo is coupled to the cycle glutamate-glutamine. Glutamine is synthesized in astrocytes from glutamate and from ammonium in an ATP dependent reaction catalysed by glutamine synthetase; glutamine leaves astrocytes and is transported into neurons, where it is hydrolyzed by a reaction catalyzed by glutaminase for the release of glutamate and ammonium. Probably the BMAA induces the use of glucose, the formation of ATP and the activity of the cycle glutamate-glutamine. Since the glutamine synthetase is inhibited by the stimulation of NMDA receptors and glutamate concentrations in the tissue and in incubation liquids were not affected by the BMAA, it is likely that the synthesis of glutamine inhibition from glutamate in brain slices is due to the activation of NMDA receptors by carbamates of BMAA (formed by bicarbonate/CO2). Carbamates are active at the receptor level AMPA at low concentrations, and on NMDA receptors at higher concentrations.

Activity on NMDA receptor

It has been demonstrated that the BMAA activates the NMDA receptor. To determine the effects of BMAA on neuronal death, toxicity induced by exposure to a concentration of BMAA equivalent to 3 mM (producing almost complete neuronal death) has been investigated and has allowed us to analyze the toxic actions of BMAA without the effects of other insults.
Previous studies (Ross, 1987; Weiss, 1989) have indicated that NMDA receptor antagonists protect against BMAA toxicity. Lobner et al. (2007) found that MK-801 (the non-competitive antagonist the NMDA receptor) blocks about 50% of the toxicity of BMAA.
The exposure to BMAA or NMDA for 1 h induced an increased influx of calcium, and the increase was largely blocked by MK-801 (blocking 88% of BMAA induced the 45Ca2 +influx, blocking 96% of NMDA induced 45Ca2 + uptake).
Therefore BMAA can cause death through the NMDA receptor, but the fact that MK-801 reduces the toxicity of BMAA of 50% has suggested the presence of additional mechanisms of toxicity.
To determine whether the effect of BMAA is directed on the NMDA receptor, cell patch-clamp electrophysiology on cultured cortical neurons has been performed, in which the BMAA, inducing a concentration-dependent current, has allowed us to measure this current at a concentration of 10 uM and to see that the current was largely mediated by the activation of NMDA receptors because the competitive antagonist of the receptor NMDA, D-amino-5-fosfonovalerato (APV), blocked 94% of the current induced by 3mM BMAA and all of the current induced by 100 uM of BMAA. The fact that the BMAA acts in a direct manner on the NMDA receptor by activating and enhancing its effect is indicated by the fact that the current induced by the toxin also existed in saturation conditions of glycine, and that the BMAA induces a significant current even in low concentrations, while MK-801 protects against its toxicity at high concentration.

Triple mechanism of neurotoxicity: activation of NMDA and mGluR5, induction of oxidative stress
The discovery of Lobner et al. β-N-methylamino-L-alanine enhances neurotoxicity through multiple mechanisms. Neurobiology of Disease 2007 that the block of NMDA receptors provides incomplete protection (50%) against the high concentrations of BMAA toxicity has led to the discovery of the involvement of other two mechanisms.
In addition to MK-801, further protection against the toxicity of BMAA have been provided also by the 6-methyl-2-[phenylethinyl]-pyridine (MPEP), mGluR5 receptor antagonist, and the free radical scavenger, Trolox. The combination of these agents has acted by distinct mechanisms, and their combination has provided a lager protection than when they are alone. The great toxicity induced by the activation of NMDA by BMAA is demonstrated also by the fact that no compound was protective in the absence of MK-801. The protection afforded by Trolox suggests that BMAA induces oxidative stress. By measuring the cellular oxidative stress with the fluorescent dye dichlorofluorescein (DCF), has been found that exposure to 3 mM BMAA for 3 hours caused a significant increase of oxidative stress, which was not attenuated by MK-801 or by MPEP, but has been blocked by Trolox, with or without MK-801.

The mechanism by which the BMAA induces the oxidative stress has not been determined but is different from activation of NMDA and mGluR5, because it is not blocked by MK-801 or MPEP. One possible mechanism for oxidative stress induced by BMAA is that it inhibits antiport the cystine/glutamate that leads to a decrease of absorption of cystine and reduction of intracellular glutathione. Despite MPEP provides protection against toxicity of BMAA in presence of MK-801, it causes increased oxidative stress. The inhibition of group I of mGluR5 increases the oxidative stress. This effect means that MPEP acts protecting against the toxicity of BMAA not inhibiting mGluR5 directly but through another mechanism, for example, the inhibition of the production of IP3 and the release of calcium from intracellular stores.
BMAA boosts the damage of some insults induced by agents such as NMDA, Fe, BSO, β-amyloid and MPP+, selectively and independently of the type of death induced by the insult (apoptosis or necrosis).
In the culture system of cortical cells, exposures to agents such as NMDA, Fe, BSO and kainate-all induced necrosis, but while the toxicity of the first three agents has been enhanced by BMAA, the kainite toxicity was not increased. The exposure to C2-ceramide and staurosporine induced apoptosis, but this was not increased by the BMAA, while the toxicity of β-amyloid and MPP + induced necrosis and apoptosis, and the damage is enhanced by BMAA. β-amyloid toxicity is considered a model of Alzheimer's disease and MPP + toxicity a model of Parkinson's disease.
The BMAA boosts the damage of all these lesions by inducing oxidative stress. Since enhances specific insults induced by other toxic agents, can increase death of cortical neurons at concentrations 100-fold lower than previously indicated. Opposite to cortical neurons, motor neurons are very sensitive to the BMAA toxicity even without an additional insult: BMAA induce the selective death of motor neurons from 30 uM. As the development of ALS / PDC seems to be caused by a long-term exposure while these studies have shown the effects of BMAA only in a short period of time (24 hours), it is likely that BMAA may increase the neuronal death even at lower concentrations of BMAA but in long periods of exposure. Most neurodegenerative diseases seem to involve excitotoxicity and oxidative stress mediated by the NMDA receptor, and activation of mGluR5 receptor was implicated in Parkinson's disease.
While previous studies on mice had shown that the administration of BMAA not induced neurological deficits or neuronal death in vivo, the results obtained from Lobner et al. β-N-methylamino-L-alanine enhances neurotoxicity through multiple mechanisms. Neurobiology of Disease 2007 have suggested that it is unlikely that the BMAA alone causes neurological deficits, unless very high concentrations of that are consumed, the combination of the consumption of low levels of BMAA with other insults (including a genetic predisposition) might it lead the development of neurodegenerative diseases, increasing the damage, accelerating the onset, or prolonging effects.


It is absolutely necessary to extend the research of BMAA to eukaryotic organisms important in food and economic life of our society that represent sources for human exposure, such as fish and filter feeders in order to check a possible transfer of the toxin in these organisms and the quantity of bioaccumulation of these through natural food chains, allowing to evaluating the actual risks of the human population.
Until 2003, the knowledge of the presence of BMAA in cycads only made think that human exposure to this neurotoxin and associated risks were limited only to tropical and sub/tropical environments where cycads grow, and to those indigenous peoples who ate products in which the BMAA is accumulated (as seed flour cycads, foxes flying). But since BMAA is distributed in the most varied environments and ecosystems of the Earth (as produced by virtually all cyanobacteria, which are ubiquitous), representing a possible threat to human health through many likely sources of exposure, it is essential conduct additional studies to show, with greater certainty, the actual role of this neurotoxin in human neurodegenerative diseases, and to clarify the possible sources of human exposure and possible risks.

This work has been made by Eleonora Virgilio and Giulia Arrigoni

2013-09-11T10:33:24 - Giulia Arrigoni
2013-09-11T10:32:44 - Giulia Arrigoni
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