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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
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Article Open Access
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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
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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
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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
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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
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 5 of 16
J Mol Genet MedISSN:1747-0862 JMGM, an open access journal
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].
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 6 of 16
J Mol Genet MedISSN:1747-0862 JMGM, an open access journal
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
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
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-
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
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
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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
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
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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
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
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Volume 9 • Issue 4 • 1000197
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-
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
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
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Volume 9 • Issue 4 • 1000197
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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.”
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
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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
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
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-
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.
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