Molecular and Genetic Medicine - People | MIT CSAILpeople.csail.mit.edu/seneff/2015/BeechamSeneff1_2015.pdfstrongly associated with agents that cause birth defects [9], and these teratogens

The Possible Link between Autism and Glyphosate Acting as Glycine Mimetic -A Review of Evidence from the Literature with AnalysisBeecham JE and Stephanie Seneff *

Computer Science and Artificial Intelligence Laboratory, Massachusetts Institute of Technology, Cambridge MA 02139, USA*Corresponding author: Stephanie Seneff, Computer Science and Artificial Intelligence Laboratory, Massachusetts Institute of Technology, Cambridge MA 02139, USA,E-mail: [email protected]

Received date: August 26, 2015, Accepted date: October 06, 2015, Published date: October 13, 2015

Copyright: © 2015 Beecham JE, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Abstract

The causes of autism spectrum disorder (ASD) are not well understood. Only a minority of cases are explainableby specific abnormalities in DNA sequence, whereas the majority are widely assumed to be linked to epigeneticeffects, and/or likely impacted by environmental factors. Here, we postulate autism causation via environmentaland/or dietary sourced toxin acting intermittently in utero on human fetuses to disrupt neurodevelopment in a non-dose dependent manner. Our theory is informed by a mini-review and correlation of selected studies from theresearch literature related to autism, including radiologic, anatomic, metabolic, neurodevelopmental, pharmacologicand MRI studies. In reviewing and analyzing evidence, we focus on data supporting interaction of the theorizedharmful glycine mimetic at one or more of the following calcium inflow regulatory factors for neurons: the N-methylD-aspartate (NMDA) receptor, the glycine receptor (GlyR) and/or the glycine transporter protein 1 (GlyT1).

We postulate this harmful glycine mimetic to act by exerting a direct molecular disruption to calcium regulatoryfactors for neurons. This disruption appears to occur in a time sensitive, rather than a strictly dose-dependentmanner, leading to haphazard disorganizations of the normally carefully choreographed steps of early neuronalmigration. Within this analysis, we find support for the contention that a strong candidate for the putative harmfulglycine mimetic is glyphosate, the active ingredient in the pervasive herbicide Roundup®. In addition to glyphosate’smolecular similarity to glycine, glyphosate is known to have a propensity to avidly bind minerals such as manganeseand magnesium, which minerals are implicated in the normal functioning of several neuronal calcium inflowregulatory factors. Our theory highlights areas deserving of further study.

IntroductionSince 1980, the number of children known to have autism spectrum

disorder (ASD) has increased dramatically. However this increase isthought to be at least partly due to changes in diagnostic practices [1].The risk of autism is known to be associated with advanced paternalage and with diabetes in the mother during pregnancy [2]. There is awell-known male preponderance in ASD cases [1]. Although autism isbelieved, by many experts, to be caused by inherited factors, no singlespecific DNA sequence alteration has been found to explain more thana minority of the cases.

A recent report by Yuen et al. [3] has raised questions about thesubject of heritable gene defects in regard to autism causation. Becauseautism often runs in families, experts had assumed that siblings withthe disorder were inheriting the same autism-predisposing genes fromtheir parents. It now appears this may not be so. These researcherssequenced, from 85 families each having two children affected byautism, 340 whole genomes, including from 170 individuals with ASD.They found that the majority of autism sibling pairs (69 percent) hadlittle to no overlap in abnormal genes. Sibling pairs shared the sameautism-associated gene changes less than one third of the time (31percent). The majority of autism-affected sibling pairs (69 percent) hadlittle to no overlap in the gene variations thought to contribute toautism.

This raises the question whether children are developing autismfrom an environmental or dietary toxin for which the DNA alterationssignal presence of the toxin, but wherein the genetic changes are notconferring all the autism-causing action. The possibility exists that theputative toxin is also, or perhaps principally, causing damage byinteracting directly within the neurodevelopmental molecularstructures and pathways of the developing brain. A candidatemolecule, as will be discussed in more detail later, is glyphosate. It isnoteworthy that, in a study by Koller et al. [4], human buccal epithelialcells from cell line TR146 were exposed to glyphosate alone in one test,and separately in another test using only the glyphosate-containingformulation known as Roundup®. Each test was at concentrationsrepresenting a 450-fold dilution of concentrations used in sprayingcrops in agriculture. Separately, in each of the two test exposures, DNAdamage was found to occur in the exposed epithelial cells.

Experts, when ascribing causation in autism, often invokeepigenetic changes, citing unspecified reactions, or perhapsenvironmental influences [5]. Such epigenetic changes, while notmodifying the DNA sequence code, are nevertheless thought to beheritable and causative, affecting early/fetal neurodevelopment [6].Environmental causes thus have not been ruled out as causative orcontributory in autism [7], and exposure to air pollution, especiallyparticulates and heavy metals, are acknowledged to perhaps increasethe risk of autism [8]. Furthermore, some cases of autism have been

Molecular and Genetic Medicine Beecham and Seneff, J Mol Genet Med 2015, 9:4http://dx.doi.org/10.4172/1747-0862.1000187Review Article Open Access

J Mol Genet MedISSN:1747-0862 JMGM, an open access journal

Volume 9 • Issue 4 • 1000197

mailto:[email protected]://dx.doi.org/10.4172/1747-0862.1000187

strongly associated with agents that cause birth defects [9], and theseteratogens are widely believed to have their greatest effect within thefirst 8 weeks from conception.

Rzhetsky et al. [10] reported in 2014 on the results of their analysisof geographic spatial incidence patterns of ASD and IntellectualDisability (ID) as reflected in insurance claims datasets representingnearly 1/3 of the entire US population. They used “the rate ofcongenital malformations of the reproductive system as a surrogate forenvironmental exposure of parents to unmeasured developmental riskfactors, including toxins” because “70 to 80% of male congenitalmalformations of the reproductive system have no clear genetic causes.Instead, they appear to be driven by specific environmental insults…”They found that, “Adjusted for gender, ethnic, socioeconomic, andgeopolitical factors, the ASD incidence rates were strongly linked topopulation-normalized rates of congenital malformations of thereproductive system in males (an increase in ASD incidence by 283%for every percent increase in incidence of malformations, 95% CI:[91%, 576%], p

that subject. To their surprise, the pattern of brain region bindingpotential was essentially the same in the brains of control subjects as inthe brains of autistic subjects; only the degree varied. In other words,in the autistic subjects the binding potential was higher, whilemaintaining the same general pattern of glial activation within brainregions as the age and IQ-matched healthy control subjects from thesame area in Japan.

In Japan, glyphosate herbicides are widely used, including for weedcontrol [14]. In addition, the country of Japan imports wheat fromcountries where glyphosate is sprayed onto wheat as a desiccant. Japanalso imports rapeseed, for use as animal feed, from countries sprayingglyphosate onto rapeseed as a desiccant. In the context of the theoryherein advanced, these findings could fit the model of anenvironmental/dietary toxin.

Selected Microscopic Studies in Autistic Human Brains

Stoner et al. [15] examined post-mortem brain tissue from 22children who died between the ages of 2 and 15. Half of the childrenstudied had been diagnosed with autism, while the other half had not.The symptoms of those with autism varied from mild to severe. In 10of the 11 autistic brains, they found patches of cortex in which thenormal pattern of gene expression and cell organization was disrupted.These areas were only a few millimeters across (roughly a quarter to ahalf inch in size) across the wide expanse of otherwise histologicallynormal-appearing cortex.

In some of the abnormal patches, a specific layer was missing. Inother patches, certain expected cells weren’t present. Similar changeswere found in 1 of the 11 children without an autism diagnosis, a childwho had suffered from seizures. All areas of cortex sampled fromautistics demonstrated such patches, however the researchers indicatedthat the most affected areas of the brain were the prefrontal cortex andthe temporal lobe cortex, while areas such as the optical cortex wererelatively spared. The researchers speculated that the defects couldhave resulted from the brain cells in autistics undergoing some sort ofdisorganization event or events during the latter part of the firsttrimester or the early part of the second trimester of fetal development.

This study by Stoner et al. left the researchers speculating aboutcausation of the observed disordered patches. They mentioned thattheir earlier studies had demonstrated that, between the ages of 2 and16 years, brains from autistic children are heavier than non-autisticchildren brains and have a relative increase in prefrontal cortex neuronnumbers of up to a startling 67%. Stoner et al. also discovered that adeficit of markers of excitatory neurons was the most robust indicatorof such a patch of disorganization. Furthermore, they did not believethis was a result of downregulation of genes. Rather, they speculatedthat the patchy areas of disorganization somehow resulted fromneurons ‘failing to reach their intended destination’ or from de novochanges in early neurodevelopment.

Lopez-Hurtado et al. [16] conducted a microscopic study of post-mortem brains from 15 age-matched autistic and control subjectsfocusing on brain regions associated with the production andprocessing of speech. Of the brains studied, 8 were from autismpatients and 7 were controls. They studied Wernicke’s area (Brodmann22, speech recognition), Broca’s area (Brodmann 44, speechproduction) and the gyrus angularis (Brodmann 39, reading) from

autistic subjects (7-44 years of age) and control subjects (8-56 years ofage). These researchers found: “Striking differences in the density ofglial cells, the density of neurons and the number of lipofuscin-containing neurons in the autistic group compared with the controlgroup. The mean density of glial cells was greater in the autistic cohortthan controls in area 22 (p

The researchers then examined brains from mice, comparingautism-model mouse neurons versus non-autism normal mouseneurons. They studied Tsc2+/- ASD mice brains. These mice havealteration in the mammalian target of rapamycin (mTOR) such thatthe mTOR is constitutively overactive. mTOR is a serine/threoninekinase, which belongs to the phosphatidylinositol-3 kinase (PI3K)related kinase (PIKKs) family. It regulates cellular metabolism, growth,and proliferation. Tang et al.’s study of such autism-model mice withconstitutively hyperactivated mTOR found signs of reduced autophagy.Autophagy is the process in neurons that, among other things, prunesneurons of excess spikes. Tang et al. [18] found that the autism-modelmice, similar to the autistic children, had too numerous spikes onneurons. They deduced that this excess presence of spikes was due to alack of adequate pruning, and postulated this was from a reduction orblockade of normal autophagy. These Tsc2+/- mice demonstratedASD-like social behaviors.

Aberrant mTOR activation resulting in reduced autophagy is knownto occur in human diseases on a genetic basis. For example, mutationsin either of 2 genes (TSC1 or TSC2) have been determined to causetuberous sclerosis complex. Among tuberous sclerosis patients, 20% to60% are also diagnosed with autism [19,20]. It has been reported that,of all autism cases, approximately 1% to 4% are tuberous sclerosispatients [21]. Treatment of tuberous sclerosis patients with rapamycincan apparently partially correct their inherited defect of reducedautophagy.

The beneficial effect of rapamycin is thought to occur viarapamycin’s action to inhibit hyperactivated mTOR and thus induce anincrease in autophagy [22]. Rapamycin has been used successfully intuberous sclerosis patients to shrink angiomyolipomas [23] andastrocytomas [24]. Clinical trials are underway to assess the feasibilityand safety of administering rapalogues sirolimus or everolimus inparticipants with Tuberous Sclerosis Complex (TSC). The assessment(Clinical Trial NCT01929642 USA) includes measuring any reductionin autistic symptoms, such as aggressive behaviors and/or anyimprovements in cognitive function.

Aberrant mTOR hyperactivation is thought to occur in a wide rangeof ASD patients with known genetic defects [25,26], such as thoseautistics found to have large head size (macrocephaly) early in life [27].This finding of large head size is reminiscent of the increased brainvolume and increased CSF accumulation found in the childrendestined to develop autism as studied by Shen et al. [12], as discussedabove. Additionally, cases of genetic disease patients linked to mTORhyperactivation who also relatively often have a diagnosis of autisminclude neurofibromatosis type I, Lhermitte-Duclos syndrome, andFragile X syndrome [21,26].

It is conceivable however, that mTOR hyperactivation can occur inautistics for which a gene sequence alteration in DNA is not present.This type of mTOR hyperactivation is theorized to occur withinneurons exposed intermittently to a toxin from diet and/orenvironment. Such a toxin is theorized herein to alter the influx ofcalcium into immature neurons in a haphazard and intermittentmanner resulting in reduction of autophagy in such neurons soexposed, with effects dependent on their stage of development.

Such a haphazard hyperactivation of mTOR has been reproducedexperimentally and linked to the presence of amino acids. Gulati et al.[28] studied HELA cells in culture and found that mTOR activationwas in relation to the level of amino acids present in the cell culture.They found that increased amino acids in the cell culture medium

resulted in an increase of HELA cell intracellular calcium, resulting inincreased calcium binding with calmodulin within the cell, andsubsequent increased activation through the mTOR pathway.Specifically, they state in their report “We demonstrate that the rise in[Ca(2+)] (i) increases the direct binding of Ca(2+)/calmodulin (CaM)to an evolutionarily conserved motif in hVps34 that is required forlipid kinase activity and increased mTOR Complex 1 signaling”.Glycine is an amino acid that is known to be present in and interactwith human brain cells, in particular during neurodevelopment. Aharmful glycine mimetic is herein theorized to be causative in at leastsome cases of autism, and to have an effect in human neurons similarto that effect of amino acids demonstrated in the cell culture studied byGuloti [28], i.e. an increased mTOR activation, thus reducingautophagy and leaving neuron spikes unpruned.

Human Post-mortem Selected Study of Autistic BrainsFinds Markers of Increased Calcium in Autistic Brainsversus Controls

Research on intracellular calcium in autism neurons offers a link toincreased mTOR activation and reduced autophagy. Palmieri et al. [29]studied temporocortical gray matter from six matched patient-controlpairs of normal and autistic brains to perform post-mortembiochemical and genetic studies of the mitochondrial aspartate/glutamate carrier (AGC). The AGC participates in the aspartate/malatereduced nicotinamide adenine dinucleotide (NAD) shuttle and isphysiologically activated by calcium (Ca(2+)). AGC transport rateswere significantly higher in tissue homogenates from all six autismpatients, including those with no history of seizures and with normalelectroencephalograms prior to death. This increase was consistentlyblunted by the Ca(2+) chelator ethylene glycol tetraacetic acid.Neocortical Ca(2+) levels were significantly higher in all six autismpatients compared to controls, according to these researchers.Furthermore, following removal of the Ca(2+)-containingpostmitochondrial supernatant, these researchers reported they thenobserved no subsequent difference in AGC transport rates in isolatedmitochondria from patients versus controls. This result clearlydemonstrates that Ca(2+) entry provoked mTOR activation, leading toreduced autophagy in association with autism.

Calcium influx relates to action at neuron membranesof glycine-related factors

An article by Avila [30] reveals the link between calcium influx intoneurons and the action of ligands at membrane-embedded proteinstructures of neurons. For example, Avila describes the glycinereceptor’s (GlyR) action as follows: “GlyR activation during embryonicand early postnatal development most likely induces a depolarizationof the cell membrane…which in turn may activate calcium influx”.

Thus, glycine binding at the GlyR appears capable of influencing therate of calcium influx into neurons during early neurodevelopment. Aswill be further explored below, we theorize action of a harmful glycinemimetic in autism causation via its influence in causing harmful andhaphazard influx of calcium into immature neurons duringneurodevelopment. To further clarify this theory, we identified andreviewed known facts and selected published literature regarding acandidate molecule widely dispersed in the environment and presentin certain foods, namely glyphosate.

Citation: Beecham JE, Seneff S (2015) The Possible Link between Autism and Glyphosate Acting as Glycine Mimetic - A Review of Evidencefrom the Literature with Analysis. J Mol Genet Med 9: 187. doi:10.4172/1747-0862.1000187

Page 4 of 16


Volume 9 • Issue 4 • 1000197

http://dx.doi.org/10.4172/1747-0862.1000187

Glyphosate, candidate glycine-mimetic molecule forcomparison to requirements of theory

Glyphosate, a common herbicide, is known to be produced from theamino acid glycine via addition of a phosphonomethyl group (Figure4). In 2007, according to the US EPA, glyphosate was the most usedherbicide in the United States agricultural sector, with 180 to 185million pounds (82,000 to 84,000 tons) applied, and the second-mostused in the home and garden market, where users applied 5 to 8million pounds (2,300 to 3,600 tons); in addition, industry, commerce,and government applied 13 to 15 million pounds (5,900 to 6,800 tons).

Figure 4: To glycine, chemist adds a phosphonomethyl group tomake glyphosate.

Glyphosate study, glyphosate from maternal diet foundconcentrated in piglet fetal brain

In a study by Kruger et al. [31] of 38 malformed piglets born to sowsfed glyphosate-containing feed, brain tissue samples were obtainedfrom euthanized 1 day old piglets’ brains. Following appropriateprocessing, these samples were tested for glyphosate using ELISA kits(Abraxis, USA). Glyphosate was found to be present in all piglet brainsamples, at an average of 3.1 micrograms glyphosate per ml of sample.The authors commented that glyphosate was able to reach the fetalpiglets from maternal feed, i.e. glyphosate from the feed eaten by thesow was able to pass the placental barrier. The feed, which contained0.87 to 1.13 parts per million glyphosate, when consumed in the first40 days of pregnancy, was associated with a rate of visible grossmalformation in the piglets of 1 per 240 piglets. No microscopicevaluation of piglet brain tissue for autistic type changes wasconducted.

Glyphosate usage on corn/soy crops versus rate of USAschool children autism

Correlation is not proof of causation, but Swanson et al. [32] pointout the near exact match in correlation between the rise of glyphosateusage on corn and soy crops in the USA, over the years 1992 to 2010,and the increase in autism rates over the same period as reported inthe USA public school system (Figure 5). Among the most widelyapplied formulations of glyphosate is the pesticide known asRoundup®. We reviewed studies of glyphosate and Roundup® in

relation to published research regarding brain tissue, including inrelation to calcium influx into neurons.

Figure 5: From Swanson et al. [32], used by permission: Near exactmatch of tons of glyphosate applied to corn/soy versus number ofchildren with autism as served under IDEA (US Department ofEducation, Individuals with Disabilities Education Act).

Glycine chelates, Glyphosate, manganese and theglutamate-glutamine cycle

Glycine, the simplest amino acid, has a molecular weight of 75, andnaturally forms chelates with cations. The strength of glycine chelatesis ideal for biologic processes, for example aiding mineral absorptionfrom the intestine, while not overly avidly binding minerals which areneeded for use in the body, such as for enzymatic reactions. Whendiscussing a putative harmful glycine mimetic in the brain, it is usefulto consider chelation in regard to the Glutamate-Glutamine cycle, andits apparent dysfunction in autism.

We begin with the synapse, the space between two neurons wherechemical signals pass. Receiving the signal are receptors at the post-synaptic neuron membrane. Among these receptors are the NMDAreceptors, which are neural membrane-embedded protein structures.When activated, the NMDA receptor opens to allow calcium to enterthe neuron. The activation process, as will be discussed in detail below,includes the simultaneous binding to the NMDA receptor of bothglutamate and glycine or a glycine mimetic [33].

Studies of autism and glutamate have documented that, in autismpatients, glutamate activity is impaired [34], both in regard to actionsat the synapse [35] where glutamate is released, and in the resultantcalcium signaling, as it relates, for example, to dendritic growth [36]. Astudy by Page et al. [37] demonstrated that the ASD patients studiedhad a significantly higher concentration of glutamate in the amygdala-hippocampal region of the brain than did normal controls. Thesefindings suggest the Glutamate-Glutamine cycle is disrupted in autism.

The cycle can be viewed as starting with the step where glutamate isreleased into the synapse by the pre-synaptic neuron. The releasedglutamate can participate in binding the NMDA receptors of the post-synaptic neuron, thus transferring a signal. Such NMDA receptoractivation is subject to other factors, as will be discussed below, but if


Page 5 of 16


Volume 9 • Issue 4 • 1000197

http://dx.doi.org/10.4172/1747-0862.1000187

the activation leads to excess calcium inflow into neurons, this can beproblematic. The Glutamate-Glutamine cycle can be viewed as one ofthe natural controls to prevent such excess calcium inflow intoneurons.

As the next step of the cycle, the free glutamate within the synapsespace is normally quickly taken up for recycling by astrocytes. Thisremoval of glutamate from the synapse helps prevent over-activation ofthe NMDA receptors. Within the astrocytes, the enzyme glutaminesynthetase (GS) normally converts glutamate to glutamine. Glutamineis then returned from the astrocytes to the neuron. Within the pre-synaptic neuron, glutamine is converted back to glutamate for storagein internal vesicles. These vesicles will later move to the pre-synapticneuron membrane and subsequently this glutamate will be releasedback into the synapse, where the cycle repeats [38].

In essence, GS can be viewed as helping protect neurons from excessglutamate activity/toxicity. However, GS activity depends onmanganese as a cofactor, and this is where a putative harmful glycinemimetic could interfere. Glyphosate, as the candidate glycine mimetic,has a well-known action as an exceptionally avid chelator of cations,including manganese. Thus, glyphosate is viewed by some experts ashaving the potential to interfere with the free supply of manganesewithin astrocytes for use by GS within the Glutamate-Glutamine cycle[39]. This is theorized as one mechanism by which a putative harmfulglycine mimetic, operating locally in patchy areas of the brain, mightcause damage to neurons by increasing glutamate to toxic levels withinthe synapse, thus spiking calcium inflow haphazardly into neurons.This theory appears to correlate to the findings of Page et al. [37]regarding excess glutamate in autistic brains in the amygdala-hippocampal area and the findings of Stoner et al. [15] regardingpatchy areas of cortical disorganization found in brains of autisticpatients.

As an example of its avidity in binding manganese and magnesium,glyphosate has been shown to deplete manganese and magnesiumlevels in young leaves of non-transgenic soybean plants exposed toglyphosate [40]. A recent study on dairy cows fed GMO Roundup-Ready feed showed dramatically reduced levels of serum manganese inassociation with glyphosate residues in the urine [41]. Autism has beenlinked to reduced manganese levels in the baby teeth of autistics [42].Seizures, which have been associated with low serum manganese[43,44], are more prevalent among children with autism [45]. We willfurther explore these matters and related factors below.

Round-up’s effects on animal brain slices - calciuminflux, apparently via NMDA receptor activation

Excess calcium entry into neurons is a well-known pathway toneuronal damage and/or disruption of neurodevelopment. In the studyconducted by Hyrc et al. [46], increasing concentrations of calciumwere applied to in vitro cultures of disassociated embryonic neuronsfrom 15 to 18 day old mice embryos. Such concentrations of calciumwere found to be predictive of neuronal death during NMDAstimulation. Cattani et al. [47] studied the effect of glyphosate onimmature neurons of the hippocampus in rats. Maternal rat exposureto the pesticide involved treating dams orally with 1% Roundup®(0.38% glyphosate) during pregnancy and lactation (until 15 days old).Hippocampal slices from 15-day-old rats were acutely exposed toRoundup(® (0.00005-0.1%) during 30 min, and experiments werecarried out to determine whether glyphosate affects (45)Ca(2+) influxand cell viability. In this preparation, these researchers investigated the

pesticide’s effects on oxidative stress parameters, (14)C-α-methyl-amino-isobutyric acid ((14)C-MeAIB) accumulation, as well asglutamate uptake, release and metabolism. They found that acuteexposure to Roundup(® (30 min) increases (45)Ca(2+) influx(apparently by activating NMDA receptors and voltage-dependentCa(2+) channels), leading to oxidative stress and neuronal cell death.Signal transduction pathways involved in the Roundup®-induced45Ca2+ uptake showed that either AP5 (a NMDA receptor antagonist)or KN-93 (a Ca2+/calmodulin-dependent protein kinase II selectiveinhibitor) prevented Roundup®-induced 45Ca2+ influx.

Cattani et al. [47] also found that such acute exposure of animalbrain slices to glyphosate increased (3)H-glutamate release into thesynaptic cleft, decreased glutathione (GSH) content and increased thelipoperoxidation, characterizing excitotoxicity and oxidative damage.They also observed that both acute and chronic exposure to Roundup(®decreased (3)H-glutamate uptake and metabolism, while inducing(45)Ca(2+) uptake and (14)C-MeAIB accumulation in immature rathippocampus.

In view of the above suggestion that glyphosate has interaction atneural membrane-embedded receptors, a review was carried outregarding structure and function of neural membrane-embeddedproteins, particularly as they relate to glycine, calcium influx andpotential sites for interaction of glyphosate and/or glycine mimetics.

Neurotransmitters and membrane-embedded proteinstructures in neurons

Glycine is widely recognized as a neurotransmitter, i.e. glycine can,under specific circumstances, have an effect on the flow of ions across aneuron’s membrane. The neuron membrane is a lipid bilayer withembedded proteins. Some of these embedded proteins are pumps, suchas the sodium-potassium ATPase pump, while many embeddedproteins are ion channels. These ion channels are transmembraneproteins with a complex configuration that includes a pore throughwhich ions can pass. Some channels permit passage of ions via openingof the pore as a result of membrane depolarization, and/or by thebinding to the membrane-embedded protein of certain chemicalligands. Most channels have selectivity; i.e. when open they allow onlycertain ions to pass.

Neural membrane-embedded proteins/receptors: Sitesfor putative harmful glycine-mimetic action

Glycine receptor (GlyR)GlyRs are formed by the assembly of five subunits which arrange

symmetrically around a central pore. The beta subunit is the only onewhich interacts with the anchoring protein gephyrin, making it key toanchoring GlyRs at the area of the synapse. Of the alpha units, fourtypes are known. Action of the GlyR, such as in terms of speed ofopening kinetics, depends on which alpha units are present within aparticular GlyR. While the alpha 1 beta combination displays thefastest kinetics, alpha 2 containing receptors are most abundant duringneurodevelopment.

GlyR’s can be activated by glycine or by taurine or alanine. Whenopen, chloride can traverse the pore, and flow is according to chloridegradient. In regard to autism, mutations in genes encoding the alpha 2subunit have been found in patients who carry a diagnosis of autism[48].


Page 6 of 16


Volume 9 • Issue 4 • 1000197

http://dx.doi.org/10.4172/1747-0862.1000187

The excellent article by Avila [30] reviews known facts about glycinereceptor activity during neurodevelopment. The brain’s early corticaldevelopment includes formation of mini-columns via migratingneurons [49]. Interneurons make up only about 15% of neurons, butare important to establishment of these first brain circuits [50,51].GlyRs have been demonstrated to have a role in controlling corticalinterneuron development [30] and migration.

A disruption of migration of neurons can thus be theorized to beinduced when GlyR activation is disrupted. In fact, abnormal halting ofneuron migration can be demonstrated by unopposed action inapplication of glycine to neurons in vitro [30]. The findings of Stoner etal. of patches of disordered clusters of cells in brains of autisticssuggests migration of neurons is abnormal in autistics. GlyRmalfunction as a result of action of a harmful glycine mimetic is hereintheorized as causative of autism in at least some cases.

Figure 6 illustrates the GlyR ligand binding site. As mentioned,research has demonstrated that besides glycine, several othermolecules can bind to the GlyR and alter the activation of GlyR. Forexample, strychnine is known to be an antagonist of the GlyR, as iscaffeine. GlyR activation has an effect on clustering of receptors atpost-synaptic sites [17].

A disruption of GlyR activation theoretically induces alterations inchloride outflow for immature neurons, and thus empowers haphazarddisruptions for calcium inflow into immature neurons via the NMDAreceptor channel (subject to occurrence of other coincident actions atthe NMDA receptor as will be discussed below). This putative harmfulglycine mimetic’s action at the GlyR, is envisioned to be via anexposure(s) from diet and/or environment, dosing the fetus or child inan intermittent variable-dose manner. The resulting haphazard calciuminfluxes theoretically enable a spectrum of severity in disrupted neuronmigration, reflective of the spectrum of symptom severity in ASD casesclinically.

Figure 6: Glycine receptor (GlyR) viewed with 5th subunit (analpha) removed.

The mechanism by which outflow of chloride from an immatureneuron affects calcium inflow into that immature neuron, has to dowith the effect of chloride outflow on the electrical charge at the

neuron’s membrane. A neural membrane usually retains a negativecharge within the cell, and a positive charge on the exterior. However,at times, such as when chloride outflow through the GlyR is high, thecharge of the membrane can change, leading to what is known asmembrane depolarization. Membrane depolarization can then alteractivity at other membrane-embedded protein structures, such as atthe NMDA receptor. Because like charges repel each other, adepolarization of the membrane, which produces a temporary positivecharge nearer the NMDA receptor, can dislodge the positively chargedmagnesium ion from its blocking position within the NMDA receptor,as illustrated in Figure 7.

Figure 7: NMDA receptor, note the magnesium ion is dislodgedfrom within the NMDA channel by the depolarization caused bychloride outflow at the GlyR when glycine binds there.

However, the NMDA receptor channel will not open to calciuminflow just because the magnesium ion exits the channel of the NMDAreceptor. The two binding sites of the NMDA receptor must also beproperly filled. A molecule capable of agonist binding at the so-calledglycine binding site of the NMDA receptor must be in place. Also, amolecule of glutamate must be bound at the glutamate binding site ofthe NMDA receptor. Only when these three events occur at the sametime, i.e. the magnesium ion’s exit from the NMDA channel, thebinding of an activating molecule to the glycine site of the NMDAreceptor, and the binding of glutamate to the glutamate site of theNMDA receptor, only then will the NMDA channel allow calcium toflow into the neuron.

From this complex set of requirements it is clear how carefullyneurodevelopment guards against haphazard or excess entry ofcalcium into immature neurons. Conversely, as theorized herein, whena harmful glycine mimetic is able to disrupt this carefullychoreographed system, the normal neurodevelopment sequence doesnot occur. Recall how calcium excess within a mouse brain model ofautism was associated with reduced autophagy and malformed poorly‘pruned’ neurons. It seems that proper neurodevelopment is criticallydependent on the correct and beneficial control of calcium entry intoimmature neurons.

Taurine, an amino acid, is also capable of binding the GlyR. Whentaurine binds to the GlyR, such taurine binding, like the binding ofglycine, opens the GlyR ion channel to gradient directed flow ofchloride. However, taurine binding appears to induce only


Page 7 of 16


Volume 9 • Issue 4 • 1000197

http://dx.doi.org/10.4172/1747-0862.1000187

approximately half as much chloride flow as compared to glycinebinding. In effect, taurine is a glycine mimetic which reduces chlorideflow compared to glycine binding of the GlyR. This implies a reducedopportunity of chloride flow to spur depolarization of the immatureneuron’s membrane. Thus, taurine is seen as a partial agonist of theGlyR.

Taurine tends to modulate the action of glycine at the GlyR, i.e.taurine’s presence can serve as a natural competition with glycine. Thisnatural competition results in fewer membrane depolarization events.In effect, taurine’s binding at the GlyR during neurodevelopment is apartial interference with glycine’s ability to boost calcium inflow eventsfor the neuron. This has profound potential to influence neuronmigration during neurodevelopment.

Because taurine, as mentioned above, only permits approximatelyhalf the chloride flow at the GlyR when compared to glycine bindingthe GlyR, it is thus less likely that a membrane depolarization willoccur with taurine bound to the GlyR (Figure 8). In fact, the aminoacid taurine is reported [52-54] to serve to counteract glutamate-induced elevations in intracellular calcium ions within neurons, andthus taurine confers protection against neurodegeneration. Theconcentration of taurine compared to the concentration of glycine inthe vicinity of the GlyR results in a choreographed competition.Taurine, in theory, will likely similarly compete for binding versusglyphosate, and therefore taurine likely would tend to protect from anytheorized harm from glyphosate binding at the GlyR.

Studies by Leon et al. [54] demonstrated a neuron-protective effectfor taurine thought to operate via modulating the calcium influx toneurons which otherwise would occur due to glutamate at the NMDAreceptor. Glutamate applied without taurine was able to driveapoptosis (neuron cell death) via release of cytochrome C. Taurineapplication along with glutamate was shown to prevent such apoptosis.

Figure 8: Taurine binding to GlyR reduces chloride flow byapproximately 50% compared to flow from glycine binding of GlyR,so no depolarization occurs with taurine bound to GlyR, thusmagnesium ion remains within NMDA receptor channel,continuing to block calcium from entering immature neuron.

The level of taurine present in the brain is known to progressivelyincrease during embryogenesis. At the same time, the different GlyRassemblies, from incorporation of different alpha subunits, means thatanother factor is present beyond taurine concentration. Different

GlyRs vary in their sensitivity to taurine. Homomeric GlyRa2, forexample, is 10 times less sensitive to taurine [30] compared to the mostsensitive GlyR.

Thus the choreography of neurodevelopment is tuned to geneticdistribution of different membrane-embedded receptors/proteins overtime, and to effects of local concentrations of competing receptoragonists. In regard to the theory herein advanced, we theorize theharmful action of a putative glycine mimetic acting at the GlyR todisrupt such delicate choreography. This damaging effect onneurodevelopment is notably a local and time-sensitive effect, ratherthan dependent solely on overall dose of toxin to the fetus or mother.

Another balanced mechanism during neurodevelopment by whichneurons and axons perform coordinated migration is the frequency ofspontaneous waves within a neuronal circuit, i.e. waves of depolarizingor ‘bursting’ activity. The effects within the brain of a disruptivedecrease of the local frequency of such spontaneous bursting activityvia toxins that act at the GlyR was demonstrated by Hansen et al.[55,56].

They examined white leghorn chick embryos’ spinal neuronmigration in ovo by cutting a square hole through the egg shell andplacing a pipette via which they administered GlyR antagonists,strychnine or picrotoxin. By thereby decreasing calcium transientinfluxes locally, and thus decreasing the normal spontaneous rhythmicbursting activity (RBA) of developing neuron circuits, they notedmigration abnormalities. Such GlyR antagonists, when administered atcertain days during gestation, also altered gene expression locally ofgenes which normally code for neuron axon guidance/adhesionproteins.

Glycine as ligand, binding to NMDA receptorAs discussed above, the NMDA receptor has a binding site for both

glutamate and glycine (D-serine also can bind the glycine site of theNMDA receptor). Only when both of these binding sites of the NMDAreceptor, the glutamate site and the glycine site, are agonistically filled,and only if the magnesium ion is simultaneously displaced from theNMDA channel, only then will the NMDA channel allow calcium toenter the neuron. As mentioned, this tight control of calcium entry hasimport for neurodevelopment. The amount of calcium entering via theneuron’s NMDA receptors is known to be further modulated by pHand zinc.

If a molecule of the herein theorized putative harmful glycinemimetic binds more avidly than glycine at the glycine binding site ofthe NMDA receptor, then the chances of calcium inflow into immatureneurons could theoretically be increased. This is because the theorizedmore avid binding of the putative harmful glycine mimetic to theNMDA receptor’s glycine site, would extend the time for thesimultaneous occurrence of the other two events, i.e. for thesimultaneous exit of the magnesium ion from the NMDA channel andthe simultaneous binding of glutamate to the NMDA receptor’sglutamate binding site.

Glycine concentration at synapse, glycine transporter proteinT1 (GlyT1)

Another mechanism by which a spectrum of severity of autism isenvisioned to occur, from the action of the putative harmful glycinemimetic, relates to glycine transport proteins. Neurotransmitterglycine participates in a recycling mechanism of uptake of glycine from


Page 8 of 16


Volume 9 • Issue 4 • 1000197

http://dx.doi.org/10.4172/1747-0862.1000187

the synapse by cells in the brain. Storage of glycine in the pre-synapticneurons occurs for a time, followed by re-release of the storedneurotransmitter back into the synapse (Figure 9).

Figure 9: Neurotransmitter glycine is taken up from synapse byGlyT1 transporter protein (not shown), stored, then re-released intosynapse to bind glycine receptor of post-synaptic neuron.

Glycine transporter proteins GlyT1 and GlyT2 are membrane-embedded proteins specific for glycine handling. The primary role ofGlyT1 is thought to be to maintain glycine concentrations belowsaturation level at postsynaptic NMDA receptors, thus modulatinginflux of calcium into neurons. GlyT1 is known to be subject toinhibition by glycine mimetics. For example, sarcosine, one of thenaturally occurring N-methyl analogues of glycine, is a knowninhibitor of GlyT1. Sarcosine is also one of the possible metabolicbreakdown products of glyphosate.

We theorize that glyphosate directly, or sarcosine from glyphosatebreakdown, act via GlyT1 inhibition to locally and intermittentlyinhibit the uptake of glycine from the synapse. We theorize that,during neurodevelopment, such inhibition of GlyT1 permits excesssynaptic glycine to accumulate. Thus glycine could bind to saturationthe NMDA receptor of an immature neuron, thus increasing calciumentry via the NMDA receptors into the immature neuron, damagingneurodevelopment.

In regard to GlyT1 inhibition and neurodevelopment, the work ofSchmitz et al. [57] is of interest. They performed unilateral intrastriatalinjection in mice of toxin 6-hydroxydopamine and studied the re-enervation response with and without GlyT1 inhibition. In the studiedmice whose glycine transporter protein1 function was inhibitedpharmacologically for 4 weeks, the axon sprouting of the neuronsresponding to the denervation was, at 7 weeks, twice as dense ascontrols. This ‘over-sprouting’ occurred via action of the NMDAreceptors, according to Schmitz et al.

If a re-enervation sequence can be conceptualized as similar to theoriginal enervation sequences of neurodevelopment, then the study ofSchmitz et al. might provide a relevant theoretical explanation. A spikeof putative harmful glycine-mimetic molecules in a localized area ofcortex in a developing human fetal brain might produce a disruptiveeffusion of neuron spikes due to local blockade of GlyT1 by the

harmful glycine mimetic (or its metabolite). Such patches of putativeover-exuberant neuron spikes are reminiscent of the findings of Tanget al. [18], and reminiscent of the increased neuron density found inpatches in brains of autistic children by Stoner et al. [15].

In fascinating studies [56,58] by Hansen et al., these researchersadministered the GlyT1 inhibitor sarcosine via in ovo method duringneurodevelopment of white leghorn chick embryos. They documentedthat sarcosine caused an increase in the frequency of calciumtransients, which translated into an increase of the frequency ofspontaneous rhythmic neuronal bursting activity. They found thisproduced abnormalities in neuron/axon migration.

In summary, Hansen et al. confirmed that rhythmic burstingactivity (RBA) of neuron circuits is key for early pathfinding decisionsby neurons. Moderate slowing of the frequency of RBA causes“motoneurons to make dorsoventral (D-V) pathfinding errors and toalter the expression of molecules involved in that decision.” Conversely,moderate speeding up of RBA in neuron circuits strongly perturbs “theanteroposterior (A-P) pathfinding process by which motoneuronsfasciculate into pool-specific fascicles at the limb base and thenselectively grow to muscle targets.” In their studies, these researchersfound that resumption of normal frequency of RBA sometimes allowedaxons to correct the A-P pathfinding errors, perhaps leaving aberrantnerves.

Aberrant connectivity has, in fact, been documented in the brains ofautistic children [59] using resting state functional magnetic resonance(fMRI) imaging. Di Martino et al., studying autistic children usingfMRI methods, concluded that their examination of functionalconnectivity (FC) of striatal networks in children with ASD revealed“abnormalities in circuits involving early developing areas, such as thebrainstem and insula, with a pattern of increased FC in ectopic circuitsthat likely reflects developmental derangement rather than immaturityof functional circuits.” This pattern also fits our theory of a dietary/environmental sourced toxin acting locally as a harmful glycinemimetic, having patchy effects in the CNS beyond the cortex.

Neuron migration has been visualized. Avila et al. [60]demonstrated in vivo, using cultured brain slices from mice embryos,that actomyosin contractions alter neuron migration. Theseresearchers recorded an example of neuron migration in video formatand have made the recording available for viewing online as movie S2of their open access report [60].

In summary, this delicately choreographed neurodevelopmentalprocess in humans, including the precise frequency of waves ofdepolarization in synchronized neurons within circuits, this pattern ofneurons which alternately sprout, prune and migrate, thischoreography can be locally disrupted, we theorize, by a relativelysmall number of molecules of a harmful glycine mimetic. From theavailable research literature, such disruption, for example via the GlyRand/or the GlyT1 and/or the NMDA receptor, can reasonably betheorized to produce neuronal/axonal migration defects reminiscent ofthose found in studies of brains of autistic patients.

Next, we will address the neuron membrane-embedded proteinstructures known as Na-K-Cl cotransporter 1 (NKCC1) and K-Clcotransporter 2 (KCC2).

Glycine, NKCC1 versus KCC2 in neurons, brain weightWithin the brain during normal neurodevelopment, the types and

numbers of membrane-embedded proteins will change as neurons


Page 9 of 16


Volume 9 • Issue 4 • 1000197

http://dx.doi.org/10.4172/1747-0862.1000187

mature. As illustrated in Figure 10, in immature neurons themembrane will typically have more NKCC1 proteins embedded, andfewer KCC2 proteins. One result is that immature neurons tend tokeep internal chloride concentration higher.

Figure 10: In normal circumstances, the membranes of immatureneurons include numerous NKCC1, fewer KCC2; intracellularchloride concentration tends to be higher in normal immatureneurons as compared to mature neurons.

As neurons proceed through normal fetal development, they willbegin to manufacture fewer of the NKCC1 proteins and insteadmanufacture more of the KCC2 proteins. Therefore, mature neuronswill have more KCC2 proteins embedded in their membrane and fewerNKCC1 proteins embedded, as illustrated in Figure 11. The result, innormal circumstances, is that mature neurons tend to have a lowerconcentration of chloride internally.

Figure 11: In normal circumstances, mature neurons have fewerNKCC1 and more KCC2; intracellular chloride concentration tendsto be lower in normal mature neurons as compared to immatureneurons.

In both the immature neuron and the mature neuron, the glycinereceptors (GlyR) aid in keeping the internal chloride concentrationwithin expected bounds (Figure 12). The programmed normal change

of neuron membrane-embedded proteins predominance, frompredominately NKCC1 (immature neurons) to predominately KCC2(mature neurons), is commonly referred to as the ‘GABA switch.’ Thisreflects the fact that gamma amino butyric acid (GABA), in normalcircumstances, acts to inhibit hyperactivity in mature neurons, such asduring birth and post-natally [61]. The GABA switch is often found tobe impaired in autism patients, a finding that we theorize might berelated to action of a harmful glycine mimetic duringneurodevelopment, as further described below.

Figure 12: Under normal circumstances, chloride flows out of animmature neuron when GlyR is open. Under normal circumstances,chloride flows into a mature neuron when GlyR is open.

Interestingly, a partial agonist of the GlyR is caffeine. It isnoteworthy that autistic children receiving a low dose of daily caffeineover weeks or months have been reported [62] to sometimesexperience improvement of autism symptoms as a result of thecaffeine.

As previously mentioned, it is well known that the brains of autisticchildren, in their early childhood years, are heavier than controls. Wepostulate that this weight increase is caused by increased water contentof the brain, such as in neuropil, glial cells and/or neurons. We believesuch water retention could be a response to excess cell-internalchloride, caused by impaired chloride exit following glyphosatebinding to glycine receptors. Chloride retention has been implicated inthe brain swelling associated with edema following brain trauma [63].Furthermore, sarcosine, a breakdown product of glyphosate, has beenshown to activate glycine receptors, but with a reduced effect on thechloride channel [64]. Hence, chloride can be expected to accumulateover time within the cell due to reduced efflux in response to sarcosineor glyphosate binding.

Glycine and the choroid plexusWith regard to cerebrospinal fluid (CSF), production is typically

balanced to resorption. The average adult produces approximately 500mL of CSF per day, but because CSF is constantly resorbed, only100-160 mL is present in adults at any one time under normalcircumstances. It has long been thought that CSF returns to thevascular system by entering the venous sinuses of the dura via thearachnoid granulations (also known as villi). However, recent research[65] has suggested that CSF flow along the cranial nerves and spinalnerve roots allows at least some CSF to flow into the brain’s lymphatic


Page 10 of 16


Volume 9 • Issue 4 • 1000197

http://dx.doi.org/10.4172/1747-0862.1000187

channels. Such flow may play a substantial role in CSF resorption, inparticular in the neonate or fetus, in which arachnoid granulations aresparsely distributed.

This apparent interplay between CSF and lymphatic fluid opens thedoor to new discoveries in brain metabolism and immune activity. Forexample, the possibility of harmful/reactive substances entering theCSF from lymph might be considered. Lymph is the fluid that batheslymph nodes, interacts with thymus-derived cells [66] and flowsthroughout the body using lymph channels. Lymph re-enters the bloodvia the lymphatic duct in the chest. These factors may be explored infuture studies to explain the immune alterations in autism.

With regard to the present theory, the precise details of glycine’saction with respect to the choroid plexus are, as yet, not well studied orunderstood. Nevertheless, the excess CSF accumulation visible on theMRI images reported by Shen et al. [12] in children destined todevelop autism is perhaps tentatively linkable to glycine, and/or atheorized harmful glycine mimetic.

For example, presence of a glycine receptor site in sheep choroidplexus was established by Preston [67]. A co-transporter of GABA andglycine known as GAT-2 has been reported within human choroidplexus cells. Studies by Schlessinger et al. [68] on the function of theGAT-2 transporter have revealed that, in addition to transportingGABA and glycine, the GAT-2 transporter also has a strong interactionwith a glycine mimetic known as glycylglycine. Thus, it is theorizedherein that a harmful glycine mimetic could act via glycine sites in thechoroid plexus to increase CSF production. The details of such actionare, as yet, unclear.

Relevant Treatment results in ASD patientsIn order to further evaluate the theory herein advanced, selected

pharmacologic treatment successes in autism are considered in light ofneural membrane-embedded proteins and neurotransmitters.

Calcium channel blockers/modulators, verapamil andketamine

In reviewing the literature regarding calcium channel blocker use intreatment of autism, no randomized clinical trials were found. Theauthors of the present article were struck, however, by an online blogseries of reports by the non-scientist father of an autistic son. Wehesitate to mention this anecdote here, but feel that even anecdotalevidence can sometimes point the way to clues of causation. Therefore,we present the story of an autistic boy, Anthony (not his real name).The father’s blog post (at Epiphanyasd.blogspot.com) indicates thatduring one summer, Anthony was dealing with allergies and wasstruggling with ‘aggressive’ symptoms of autism, as per his father’sreports online. As the blog describes, Anthony was saying ‘Be nice’ and‘to hit your head’ associated with attempts to self-control his flares ofaggressive autistic symptoms.

The father reports giving Anthony an anti-histamine during thatsummer, but found it was only effective for Anthony for a couple ofhours per dose. The father, as he mentioned in his posts, hadresearched the work of Italian professor and clinician Antonio Persico[69] in regards to calcium and the autistic brain. The father reports heresearched the calcium channel blocker verapamil. Satisfied that a lowdose of verapamil would be safe for his son, and seeing Anthonycontinuing to demonstrate aggressive symptoms, the father beganAnthony on a dose of 20 milligrams verapamil. The father recorded

online the circumstances regarding his son as follows: “One afternoon,I decided to give a very small dose (20 mg) of Verapamil, and beforemy eyes, the anger and agitation began to fade and was replaced bycalm. It was the most amazing experiment that I have witnessed andwithin 20 minutes there was complete calm”.

While anecdotal reports clearly have obvious limits, they maysometimes serve as a clue to areas deserving of further study underapplication of a validated scientific method. In addition, although theauthors of the present article do not recommend that parentsindependently apply non-prescribed medical regimens for theirautistic children, it is heartening to read the follow-up note in the blogconcerning Anthony. As the father described it: “In the followingweeks, I would still hear Anthony say `be nice,’ but this was no longerfollowed by any aggressive behavior. The trigger was still there toenergize these channels, but they had been blocked by Verapamil. Itwas like firing a gun, but with no ammunition; there was a `click,’ butno `bang.’ “

Another calcium channel blocker, ketamine, has been reported inthe scientific literature to have value in treating autism. Ketamine is aknown antagonist of the NMDA receptor, capable of reducing theinflux of calcium into neurons. Successful anesthesia of 5 autisticchildren has been reported by an anesthesiology group using oralketamine preoperatively [70]. The anesthesiology group indicated thatthey found the symptoms of autism were less likely to interfere withthe surgery if the patient was given ketamine orally in the preoperativeperiod. This report of ketamine success in reducing symptoms ofautism appears to link reduction of calcium entry into neurons withreduction in autism symptoms.

NKCC1 Chloride channel and bumetanideIn the brain, the diuretic bumetanide blocks action of the

membrane embedded protein NKCC1, and thus can alter internalchloride concentration within neurons. Bumetanide, when given topregnant mice with models of autism, has been reported to prevent orreduce autistic behavior in offspring [61]. In humans, Lemonnier et al.[71] reported successful use of bumetanide in a 10 month treatmentstudy in adolescents/young adults with autism. These researchersstated that their treatment was based on their understanding thatbumetanide decreases the level of intra-neuronal chloride (Cl-)i andreinforces the natural GABA inhibition of excitation of neurons inautistic patients. They report reduced severity of autism symptoms andindicate that bumetanide treatment improves emotion recognition andenhances the activation of brain regions involved in social andemotional perception during the perception of emotional faces bybumetanide-treated autistics.

The theory herein advanced proposes the harmful action of aputative glycine mimetic to over-concentrate chloride within immatureneurons. Bumetanide, by blocking such chloride-concentrating action,might at least temporarily relieve such excess chloride concentrationswithin immature neurons.

Glutamate and the Multi-system nature of autism, foodchoices linked to putative harmful glycine-mimetic

Certain chemistry findings are typical of autistic patients, such aselevated plasma and brain glutamate [72] and an altered amino acidprofile in the blood [73]. In fact, autism is widely recognized to be amulti-system disorder [74]. For example, pediatricians have described


Page 11 of 16


Volume 9 • Issue 4 • 1000197

http://dx.doi.org/10.4172/1747-0862.1000187

in autistics a somewhat characteristic pattern of signs and symptomsincluding smelly bowel movements, bloated bellies, dry skin andfrequent colds and ear infections. Such multi-system manifestations inautism will be very briefly addressed below to indicate their linkage,within the theory herein advanced, to a putative causative harmfulglycine mimetic.

Modern processed food is well known to contain excess ammoniadue to the methods of food processing. For example, as far back as1973, elevated ammonia content of gelatin, cheese, breakfast cereal,bacon, corn, peas, numerous other vegetables, cheddar cheese,buttermilk and other foods was reported in the scientific literature[75]. Injecting ammonia into meat as a means to control E. coli hasbeen accepted practice for years, though only recently widelypublicized. Part of the reason the public was not generally aware of theelevated ammonia content of hamburger and what is known as ‘pinkslime’ as a meat component, was that the amount of ammonia usedwas not required to be listed as an ingredient on the label due to thefact that the government viewed the ammonia as a processingchemical.

Under normal circumstances, excess ammonia would be a burdenfor normal human digestion. We theorize this burden is made moredifficult by the presence of a harmful glycine mimetic within such highammonia food. We theorize that the combination in food of excessammonia and a harmful glycine mimetic having biocide capabilityalters the gut microbiome, the gut lining, the blood chemistry, and hasimplications for neurodevelopment.

In order to better describe this theory, it is instructive to begin byreviewing the findings of a study of gobie fish by Peh et al. [76]. Theystudied ammonia detoxification in the gobie fish euryhalineBostrychus sinensis exposed to excess ammonia in a hyperosmoticenvironment, whereby drinking was essential for osmoregulation. Theyfound alterations in intestine/contents, which alterations they linked toaction of the enzyme system the fish uses to de-toxify ammonia. Thisenzyme system is the well-known glutamate dehydrogenase (GDH) -glutamine synthetase (GS) system. The expected products of the GDH-GS system in detoxifying ammonia include glutamine and glutamate,with perhaps un-detoxified ammonia left over if the ammoniasubstrate was in excess.

In their study of the gobie fish, Peh et al. did find in intestine/contents a significantly elevated glutamine level after ammoniaexposure, a sign the GDH-GS system was in operation and detoxifyingammonia. But they did not find elevated glutamate in the intestine/contents. They reasoned that the glutamate must have been generatedfrom the GDH-GS system, but had apparently been absorbed into theblood stream of the fish, likely so that this relatively toxic glutamatecould be either detoxified within other organs, or changed into otheramino acids.

Turning now to studies in autistics, Aldred et al. [73] studied bloodsamples from patients with autism or Asperger syndrome and bloodsamples from their siblings and parents. They found that all familymembers had the same pattern, with raised glutamate levels in theirplasma, but reduced plasma glutamine. This is reminiscent of thepattern of glutamine in the intestine and glutamate absorbed into thecirculation from ammonia detoxification as found by Peh et al. in thegobie fish. This pattern in the autism families studied by Aldred et al.,we believe, is due to the detoxification of excess dietary ammonia bytheir human gut microbiome.

But, autistics it seems, also have other markers suggestingdetoxification of excess food ammonia. In addition to the decreasedplasma glutamine and increased plasma glutamate, Aldred et al. alsomeasured plasma levels of other amino acids. They found autisticpatients and their family members had elevated plasma levels ofalanine, phenylalanine, asparagine, tyrosine and lysine when comparedto controls.

These findings suggest to us that autistics and their family memberswere eating, generally speaking, a very similar diet of high-ammoniafoods as compared to controls, food that also putatively contained aharmful glycine mimetic. This pattern of plasma amino acid elevationsin autistics and their family members, we believe, fits the pattern ofexcess food ammonia with detoxification via GDH-GS. Our view isthat some of the excess glutamate absorbed from the intestine of theautistics and the intestine of their family members became substrate.We believe, for example, some glutamate was transaminated by alanineaminotransferase into lactate-derived pyruvate to form alanine.Elevated plasma alanine was found by Aldred et al. [73] in autistics andtheir family members.

Autism, gut microbiotaDamage to neurodevelopment within the fetus is herein theorized

from maternal exposure to a putative harmful glycine mimetic.However, neurodevelopment continues after birth. This raises thepossibility of damage to developing neurons occurring from a harmfulglycine mimetic in the child after birth. For example, once thenewborn begins developing his or her own gut microflora, exposure toa harmful glycine mimetic with biocide capabilities could alter gutmicroflora, contributing to excess glutamate in blood and in the brainsof autistics if excess ammonia is present. Gut flora alterations havebeen well documented in autism [77-79], as well as leaky gut syndrome[80,81], excess fecal content of short chain fatty acids and elevatedammonia [82]. In vivo studies on rats have shown that ammoniaadministered via injection of ammonium acetate activates NMDAreceptors in the brain, leading to increased intracellular calcium,calcium uptake into mitochondria, and initiation of neuronal death[83].

Regarding gut microbiota, the excellent review and analysis byKrajmalnik-Brown et al. (65) found a ‘hyper-Westernization’ of the gutmicrobiota of children with ASD. They speculated this alteration“could indicate that gut microbiota differences that are driven byunique aspects of the Western lifestyle compared to the developingworld lead to the association of unique gut microbiota compositionwith ASD.” If indeed it is present, the nature of that unique microbiotacomposition in ASD still eludes science. As these researchersconcluded, “The complexity of the symptoms and the etiology of ASDcoupled with the complexity of the microbiota and its functions haspresented challenges in establishing the nature of an associationbetween gut microbiota and ASD, pinning down whether a link evenexists and for which individuals with ASD, and in producing amechanistic understanding of the nature of this association.”

Some of the reported changes in gut flora in autistics are thought tobe related to frequent use in autistics of antibiotics, such as intreatment of otitis media. However, studies [84,85] have so far foundno significant difference in gut microbiome between autistics and theirneurotypical non-autistic siblings. As one study concluded, “Resultsdid not indicate clinically meaningful differences between groups.”


Page 12 of 16


Volume 9 • Issue 4 • 1000197

http://dx.doi.org/10.4172/1747-0862.1000187

Nevertheless, glyphosate does appear to have the capability to alterthe gut microbiome, Shehata et al. [79] tested bacteria in culturemedium against various strengths of herbicide-formulated glyphosate.They found that most of the pathogenic bacteria they tested wereresistant to glyphosate, while most of the beneficial bacteria werefound to be moderately to highly susceptible. Thus, glyphosate appearsto have a tendency to reduce beneficial species, perhaps contributing tothe hyper-Westernized gut microflora described by researchersstudying the subject of gut microflora in autism.

There are studies which hint at differences in gut microbiotabetween autistics and non-autistic non-siblings. Finegold et al. [78] in2002 reported on a study of gastric, small bowel and stool samplesfrom late onset autism patients compared to non-sibling controls.Special care was taken to capture and culture anaerobes. Many of theseautism patients were on a gluten-free, casein-free diet. All hadgastrointestinal symptoms, primarily diarrhea and/or constipation. Allpatients had received no antibacterial agents for at least 1 month priorto the study. Results indicated a definite difference between groups, i.e.“Children with autism had 9 species of Clostridium not found incontrols, whereas controls yielded only 3 species not found in childrenwith autism. In all, there were 25 different clostridial species found. Ingastric and duodenal specimens; the most striking finding was totalabsence of non-spore-forming anaerobes and microaerophilic bacteriafrom control children and significant numbers of such bacteria fromchildren with autism.”

Limitations of study design in earlier autism researchAutism appears to be associated with relatively minor and difficult

to visualize histologic manifestations, such as patchy disruptions ofmini-column organization in brain development [86,87], as discussedabove. Studies focused on larger scale defects in neurodevelopmentsuch as malformations may miss such key localized findings. A studyof sub-lethal dosing of rats with glyphosate [88] did documentvacuolar changes in brain tissue but claimed to find no other changes,without referencing neuronal clustering or orderliness of mini-columnarrangement of neurons or the like. In order to answer the question ofwhether a putative glycine-mimetic candidate molecule, such asglyphosate, might play a role in autism causation, it will be useful todesign studies which have the potential to shed light on the relevantissues.

Risperidone, NMDA receptors, AMPA receptorsThe first drug approved by the FDA for treatment of autism,

risperidone, is the most widely used. Risperidone can have severe sideeffects, but can also prove effective in reducing tantrums, aggressionand self-injury. The improvement is generally seen in approximatelyhalf the patients and can be dramatic, taking effect in a matter ofweeks. Symptoms often return, however, when the drug isdiscontinued. Side effects of weight gain, sleepiness and high prolactineffects limit the use of risperidone.

For the purpose of the theory herein advanced, the mechanism ofaction of risperidone is relevant. Current theories of action tend tofocus on risperidone and blockage of D2 and 5-HT2A receptors;however, the study by Choi et al. [89] suggests an action of risperidonein regard to other neuron receptors. These researchers compared 3weeks of dosing at 3 different levels of risperidone in juvenile rats ascompared to the same dosing in adults rats. They found “Risperidone(at 1.0 and 3.0 mg/kg/day) significantly decreased NMDA binding in

caudate-putamen of juvenile and adult animals.” In contrast, the sametwo doses of risperidone “decreased NMDA receptors in nucleusaccumbens of juveniles and not adults.”

They also found that “risperidone (at 1.0 and 3.0 mg/kg/day)increased AMPA receptors in medial prefrontal cortex and caudate-putamen of juvenile animals, whereas risperidone (at 3.0 mg/kg)increased AMPA receptors in caudate-putamen and hippocampus ofadults.” These findings fit the theory herein advanced that haphazardcalcium inflow into neurons is key to autism. For example, suchhaphazard calcium inflow should theoretically be reduced whenNMDA receptor activity is decreased. Risperidone success, in thestudy, was associated with a reduction of NMDA receptors. Also, theincrease of AMPA receptors documented in the Choi et al. study ofrisperidone mechanism of action fits the theory herein advanced,because AMPA receptors within the brain are generally of theGluR2(R) type. The GluR2(R) receptor is known to admit sodium andpotassium, while not permitting calcium to inflow.

Discussion

The available evidence appears to link research findings in autism todefects in early (including in utero) neurodevelopment [15]. Scientificstudies are still ongoing to determine which governing factors forhuman neurodevelopment act at which steps to influence neuronmigration and/or gene expression. However, in view of the risingincidence of autism diagnosis, we hold that it is time to consider thetheory herein advanced that human fetal neurodevelopment might bealtered by exposure to intermittent doses of a relatively small numberof molecules of a putative harmful glycine-mimetic.

The theory that such a toxin is originating from environmentaland/or dietary sources is supported by the finding that the brains of atleast some of the control subjects in the studies by Suzuki et al. [13]and Stoner et al. [15] had findings similar to the findings in the brainsof autistics. Similarly, the plasma amino acid changes documented byAldred et al. [73] in autistics were present also in their familymembers. Similarly, the fact that studies of gut microbiota of autisticscompared to gut microbiota of their non-autistic siblings [84] have notshown significant differences could be explained by the presence infood for both groups of a harmful glycine mimetic acting as a biocide.

Importantly, the theory herein advanced holds that anenvironmental or dietary sourced exposure to a human fetus of such aputative harmful glycine mimetic would likely not follow a standarddose response curve. We believe evidence [30] supports the view thatthe effects on neurodevelopment in the fetus of such theorized harmfulglycine mimetic exposure are dependent on factors such as timing ofexposure, and on location of molecular interactions. This theory, ifproven, might have far reaching implications, such as precluding, atleast for pregnant women, the supposed utility of safe allowable limitsin food or on crops of the putative harmful glycine mimetic.

In essence, neurodevelopment is unlike other natural processes, inthat a small number of molecules disrupting neurodevelopment at justthe wrong time could have devastating autism-causing and long lastingoutsized effects [9]. This viewpoint is valid, we believe, because fetalneurodevelopment is not a steady state process [9]. Rather, fetalneurodevelopment, especially in its early stages, has delicate andnaturally choreographed steps [9,30] which appear inordinatelysensitive to small disruptive insults. Such small disruptive intermittentevents coming from a harmful glycine mimetic during


Page 13 of 16


Volume 9 • Issue 4 • 1000197

http://dx.doi.org/10.4172/1747-0862.1000187

neurodevelopment could explain previously puzzling autism featuressuch as the patchy cortical foci of disorganization [15] or the autisticbrain’s functional connectivity alterations, or the failure of properdevelopment of the GABA switch [61], or the wide spectrum ofsymptoms in ASD.

We recognize that this paper is speculative, but we hope it willinspire others to conduct research to test the validity of our proposedhypothesis. If our ideas are validated, it is imperative for governmentsto take regulatory action against the practice of widespread glyphosateusage on food crops.

AcknowledgementThis research is supported in part by Quanta Computers, Taiwan,

under the auspices of the Qmulus program.

References1. Newschaffer CJ, Croen LA, Daniels J, Giarelli E, Grether JK, et al. (2007)

The epidemiology of autism spectrum disorders. Annu Rev Public Health28: 235-258.

2. Gardener H, Spiegelman D, Buka SL (2009) Prenatal risk factors forautism: comprehensive meta-analysis. Br J Psychiatry 195: 7-14.

3. Yuen RK, Thiruvahindrapuram B, Merico D, Walker S, Tammimies K, etal. (2015) Whole-genome sequencing of quartet families with autismspectrum disorder. Nat Med 21: 185-191.

4. Koller VJ, Furhacker M, Nersesyan A, MiÅ¡Ãk M, Eisenbauer M, et al.(2012) Cytotoxic and DNA-damaging properties of glyphosate andRoundup in human-derived buccal epithelial cells. Arch Toxicol 86:805-813.

5. Freitag CM (2007) The genetics of autistic disorders and its clinicalrelevance: a review of the literature. Mol Psychiatry 12: 2-22.

6. Rapin I, Tuchman RF (2008) Autism: definition, neurobiology, screening,diagnosis. Pediatr Clin North Am 55: 1129-1146, viii.

7. Lyall K, Schmidt RJ, Hertz-Picciotto I (2014) Maternal lifestyle andenvironmental risk factors for autism spectrum disorders. Int J Epidemiol43: 443-464.

8. Rutter M (2005) Incidence of autism spectrum disorders: changes overtime and their meaning. Acta Paediatr 94: 2-15.

9. Arndt TL, Stodgell CJ, Rodier PM (2005) The teratology of autism. Int JDev Neurosci 23: 189-199.

10. Rzhetsky A, Bagley SC, Wang K, Lyttle CS, Cook EH Jr, et al. (2014)Environmental and state-level regulatory factors affect the incidence ofautism and intellectual disability. PLoS Comput Biol 10: e1003518.

11. Haar S, Berman S, Behrmann M, Dinstein I (2014) AnatomicalAbnormalities in Autism? Cereb Cortex.

12. Shen MD, Nordahl CW, Young GS, Woolton-Gorges SL, Lee A, et al.(2013) Early brain enlargement and elevated extra-axial fluid in infantswho develop autism spectrum disorder. Brain 136: 2825-2835.

13. Suzuki K, Sugihara G, Ouchi Y, Nakamura K, Futatsubashi M, et al.(2013) Microglial activation in young adults with autism spectrumdisorder. JAMA Psychiatry 70: 49-58.

14. Niinomi Y, Ikeda M, Yamashita M, Ishida Y, Asai M, et al. (2013)Glyphosate-resistant Italian ryegrass (Lolium multiflorum) on rice paddylevees in Japan Weed Biology and Management 13: 31-38.

15. Stoner R, Chow ML, Boyle MP, Sunkin SM, Mouton PR, et al. (2014)Patches of disorganization in the neocortex of children with autism. NEngl J Med 370: 1209-1219.

16. Lopez-Hurtado E, Prieto J J (2008) A Microscopic Study of Language-Related Cortex in Autism Am J Biochem Biotech 4(2): 130-45.

17. Kirsch J, Betz H (1998) Glycine-receptor activation is required forreceptor clustering in spinal neurons. Nature 392: 717-720.

18. Tang G, Gudsnuk K, Kuo SH, Cotrina ML, Rosoklija G, et al. (2014) Lossof mTOR-dependent macroautophagy causes autistic-like synapticpruning deficits. Neuron 83: 1131-1143.

19. Smalley SL (1998) Autism and tuberous sclerosis. J Autism Dev Disord28: 407-414.

20. Bolton PF, Park RJ, Higgins JN, Griffiths PD, Pickles A (2002) Neuro-epileptic determinants of autism spectrum disorders in tuberous sclerosiscomplex. Brain 125: 1247-1255.

21. Fombonne E (2003) Epidemiological surveys of autism and otherpervasive developmental disorders: an update. J Autism Dev Disord 33:365-382.

22. Johnson SC, Rabinovitch PS, Kaeberlein M (2013) mTOR is a keymodulator of ageing and age-related disease. Nature 493: 338-345.

23. Wienecke R, Fackler I, Linsenmaier U, Mayer K, Licht T, et al. (2006)Antitumoral activity of rapamycin in renal angiomyolipoma associatedwith tuberous sclerosis complex. Am J Kidney Dis 48: e27-29.

24. Franz DN, Leonard J, Tudor C, Chuck G, Care M, et al. (2006)Rapamycin causes regression of astrocytomas in tuberous sclerosiscomplex. Ann Neurol 59: 490-498.

25. Neves-Pereira M, Müller B, Massie D, Williams JH, O'Brien PC, et al.(2009) Deregulation of EIF4E: a novel mechanism for autism. J MedGenet 46: 759-765.

26. Ehninger D, Silva AJ (2011) Rapamycin for treating Tuberous sclerosisand Autism spectrum disorders. Trends Mol Med 17: 78-87.

27. Butler MG, Dasouki MJ, Zhou XP, Talebizadeh Z, Brown M, et al. (2005)Subset of individuals with autism spectrum disorders and extrememacrocephaly associated with germline PTEN tumour suppressor genemutations. J Med Genet 42: 318-321.

28. Gulati P, Gaspers LD, Dann SG, Joaquin M, Nobukuni T, et al. (2008)Amino acids activate mTOR complex 1 via Ca2+/CaM signaling tohVps34. Cell Metab 7: 456-465.

29. Palmieri L, Papaleo V, Porcelli V, Scarcia P, Gaita L, et al. (2010) Alteredcalcium homeostasis in autism-spectrum disorders: evidence frombiochemical and genetic studies of the mitochondrial aspartate/glutamatecarrier AGC1. Mol Psychiatry 15: 38-52.

30. Avila A, Nguyen L, Rigo JM (2013) Glycine receptors and braindevelopment. Front Cell Neurosci 7: 184.

31. Kruger M, Schrödl W, Pedersen Ib, Shehata AA (2014) Detection ofGlyphosate in Malformed Piglets. J Environ Anal Toxicol 4: 230.

32. Swanson NL, Leu A, Abrahamson J, Wallet B (2014) Geneticallyengineered crops, glyphosate and the deterioration of health in theUnited States of America. Journal of Organic Systems 9.

33. Laube B, Kuryatov A, Kuhse J, Betz H (1993) Glycine-glutamateinteractions at the NMDA receptor: role of cysteine residues. FEBS Lett335: 331-334.

34. Choudhury PR, Lahiri S, Rajamma U (2012) Glutamate mediatedsignaling in the pathophysiology of autism spectrum disorders.Pharmacol Biochem Behav 100: 841-849.

35. Cuscó I, Medrano A, Gener B, Vilardell M, Gallastegui F, et al. (2009)Autism-specific copy number variants further implicate thephosphatidylinositol signaling pathway and the glutamatergic synapse inthe etiology of the disorder. Hum Mol Genet 18: 1795-1804.

36. Krey JF, Dolmetsch RE (2007) Molecular mechanisms of autism: apossible role for Ca2+ signaling. Curr Opin Neurobiol 17: 112-119.

37. Page LA, Daly E, Schmitz N, Simmons A, Toal F, et al. (2006) In vivo 1H-magnetic resonance spectroscopy study of amygdala-hippocampal andparietal regions in autism. Am J Psychiatry 163: 2189-2192.

38. Stobart JL, Anderson CM (2013) Multifunctional role of astrocytes asgatekeepers of neuronal energy supply. Front Cell Neurosci 7: 38.

39. Samsel A, Seneff S (2015) Glyphosate, pathways to modern diseases III:Manganese, neurological diseases, and associated pathologies. SurgNeurol Int 6: 45.

40. Cakmak, I, Yazici A, Tutus Y, Ozturk L (2009) Glyphosate reduced seedand leaf concentrations of calcium, manganese, magnesium, and iron innon-glyphosate resistant soybean. Eur Journal of Agronomy 31: 114-119.


Page 14 of 16


Volume 9 • Issue 4 • 1000197

http://www.ncbi.nlm.nih.gov/pubmed/17367287http://www.ncbi.nlm.nih.gov/pubmed/17367287http://www.ncbi.nlm.nih.gov/pubmed/17367287http://www.ncbi.nlm.nih.gov/pubmed/19567888http://www.ncbi.nlm.nih.gov/pubmed/19567888http://www.ncbi.nlm.nih.gov/pubmed/25621899http://www.ncbi.nlm.nih.gov/pubmed/25621899http://www.ncbi.nlm.nih.gov/pubmed/25621899http://www.ncbi.nlm.nih.gov/pubmed/22331240http://www.ncbi.nlm.nih.gov/pubmed/22331240http://www.ncbi.nlm.nih.gov/pubmed/22331240http://www.ncbi.nlm.nih.gov/pubmed/22331240http://www.ncbi.nlm.nih.gov/pubmed/17033636http://www.ncbi.nlm.nih.gov/pubmed/17033636http://www.ncbi.nlm.nih.gov/pubmed/18929056http://www.ncbi.nlm.nih.gov/pubmed/18929056http://www.ncbi.nlm.nih.gov/pubmed/24518932http://www.ncbi.nlm.nih.gov/pubmed/24518932http://www.ncbi.nlm.nih.gov/pubmed/24518932http://www.ncbi.nlm.nih.gov/pubmed/15858952http://www.ncbi.nlm.nih.gov/pubmed/15858952http://www.ncbi.nlm.nih.gov/pubmed/15749245http://www.ncbi.nlm.nih.gov/pubmed/15749245http://www.ncbi.nlm.nih.gov/pubmed/24625521http://www.ncbi.nlm.nih.gov/pubmed/24625521http://www.ncbi.nlm.nih.gov/pubmed/24625521http://www.ncbi.nlm.nih.gov/pubmed/25316335http://www.ncbi.nlm.nih.gov/pubmed/25316335http://www.ncbi.nlm.nih.gov/pubmed/23404112http://www.ncbi.nlm.nih.gov/pubmed/23404112http://www.ncbi.nlm.nih.gov/pubmed/23404112http://www.ncbi.nlm.nih.gov/pubmed/24670167http://www.ncbi.nlm.nih.gov/pubmed/24670167http://www.ncbi.nlm.nih.gov/pubmed/24670167http://www.ncbi.nlm.nih.gov/pubmed/9565032http://www.ncbi.nlm.nih.gov/pubmed/9565032http://www.ncbi.nlm.nih.gov/pubmed/25155956http://www.ncbi.nlm.nih.gov/pubmed/25155956http://www.ncbi.nlm.nih.gov/pubmed/25155956http://www.ncbi.nlm.nih.gov/pubmed/9813776http://www.ncbi.nlm.nih.gov/pubmed/9813776http://www.ncbi.nlm.nih.gov/pubmed/12023313http://www.ncbi.nlm.nih.gov/pubmed/12023313http://www.ncbi.nlm.nih.gov/pubmed/12023313http://www.ncbi.nlm.nih.gov/pubmed/12959416http://www.ncbi.nlm.nih.gov/pubmed/12959416http://www.ncbi.nlm.nih.gov/pubmed/12959416http://www.ncbi.nlm.nih.gov/pubmed/23325216http://www.ncbi.nlm.nih.gov/pubmed/23325216http://www.ncbi.nlm.nih.gov/pubmed/16931204http://www.ncbi.nlm.nih.gov/pubmed/16931204http://www.ncbi.nlm.nih.gov/pubmed/16931204http://www.ncbi.nlm.nih.gov/pubmed/16453317http://www.ncbi.nlm.nih.gov/pubmed/16453317http://www.ncbi.nlm.nih.gov/pubmed/16453317http://www.ncbi.nlm.nih.gov/pubmed/19556253http://www.ncbi.nlm.nih.gov/pubmed/19556253http://www.ncbi.nlm.nih.gov/pubmed/19556253http://www.ncbi.nlm.nih.gov/pubmed/21115397http://www.ncbi.nlm.nih.gov/pubmed/21115397http://www.ncbi.nlm.nih.gov/pubmed/15805158http://www.ncbi.nlm.nih.gov/pubmed/15805158http://www.ncbi.nlm.nih.gov/pubmed/15805158http://www.ncbi.nlm.nih.gov/pubmed/15805158http://www.ncbi.nlm.nih.gov/pubmed/18460336http://www.ncbi.nlm.nih.gov/pubmed/18460336http://www.ncbi.nlm.nih.gov/pubmed/18460336http://www.ncbi.nlm.nih.gov/pubmed/18607376http://www.ncbi.nlm.nih.gov/pubmed/18607376http://www.ncbi.nlm.nih.gov/pubmed/18607376http://www.ncbi.nlm.nih.gov/pubmed/18607376http://www.ncbi.nlm.nih.gov/pubmed/24155690http://www.ncbi.nlm.nih.gov/pubmed/24155690http://www.ncbi.nlm.nih.gov/pubmed/7903251http://www.ncbi.nlm.nih.gov/pubmed/7903251http://www.ncbi.nlm.nih.gov/pubmed/7903251http://www.ncbi.nlm.nih.gov/pubmed/21756930http://www.ncbi.nlm.nih.gov/pubmed/21756930http://www.ncbi.nlm.nih.gov/pubmed/21756930http://www.ncbi.nlm.nih.gov/pubmed/19246517http://www.ncbi.nlm.nih.gov/pubmed/19246517http://www.ncbi.nlm.nih.gov/pubmed/19246517http://www.ncbi.nlm.nih.gov/pubmed/19246517http://www.ncbi.nlm.nih.gov/pubmed/17275285http://www.ncbi.nlm.nih.gov/pubmed/17275285http://www.ncbi.nlm.nih.gov/pubmed/17151175http://www.ncbi.nlm.nih.gov/pubmed/17151175http://www.ncbi.nlm.nih.gov/pubmed/17151175http://www.ncbi.nlm.nih.gov/pubmed/23596393http://www.ncbi.nlm.nih.gov/pubmed/23596393http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4392553/http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4392553/http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4392553/http://dx.doi.org/10.4172/1747-0862.1000187

41. Krüger M, Schrodl W, Neuhaus J, Shehata AA (2013) Field investigationsof glyphosate in urine of Danish dairy cows J Environ Anal Toxicol 3: 1-7.

42. Abdullah MM, Ly AR, Goldberg WA, Clarke-Stewart KA, Dudgeon JV, etal. (2012) Heavy metal in children's tooth enamel: related to autism anddisruptive behaviors? J Autism Dev Disord 42: 929-936.

43. Carl GF, Keen CL, Gallagher BB, Clegg MS, Littleton WH, et al. (1986)Association of low blood manganese concentrations with epilepsy.Neurology 36: 1584-1587.

44. Sohler A, Pfeiffer CC (1979) A direct method for the determination ofmanganese in whole blood. Patients with seizure activity have low bloodlevels. J Orthomol Psych 8: 275-280.

45. Volkmar FR, Nelson DS (1990) Seizure disorders in autism. J Am AcadChild Adolesc Psychiatry 29: 127-129.

46. Hyrc K, Handran SD, Rothman SM, Goldberg MP (1997) Ionizedintracellular calcium concentration predicts excitotoxic neuronal death:observations with low-affinity fluorescent calcium indicators. J Neurosci17: 6669-6677.

47. Cattani D, de Liz Oliveira Cavalli VL, Heinz Rieg CE, Domingues JT, Dal-Cim T, et al. (2014) Mechanisms underlying the neurotoxicity induced byglyphosate-based herbicide in immature rat hippocampus: involvementof glutamate excitotoxicity. Toxicology 320: 34-45.

48. Piton A, Gauthier J, Hamdan FF, Lafrenière RG, Yang Y, et al. (2011)Systematic resequencing of X-chromosome synaptic genes in autismspectrum disorder and schizophrenia. Mol Psychiatry 16: 867-880.

49. Dupont E, Hanganu IL, Kilb W, Hirsch S, Luhmann HJ (2005) Rapiddevelopmental switch in the mechanisms driving early cortical columnarnetworks. Nature 439: 79-83.

50. Cossart R (2011) The maturation of cortical interneuron diversity: howmultiple developmental journeys shape the emergence of proper networkfunction? Curr Poin Neurobiol 21: 160-168.

51. Sauer JF, Bartos M (2011) Postnatal differentiation of cortical interneuronsignalling. Eur J Neurosci 34: 1687-1696.

52. Wu H, Jin Y, Wei J, Jin H, Sha D, et al. (2005) Mode of action of taurine asa neuroprotector. Brain Res 1038: 123-131.

53. Wu JY, Wu H, Jin Y, Wei J, Sha D,

Documents

Molecular and Genetic Medicine - People | MIT CSAILpeople.csail.mit.edu/seneff/2015/BeechamSeneff1_2015.pdfstrongly associated with agents that cause birth defects [9], and these teratogens