Review Acta Neurobiol Exp 2012, 72: 113–153
© 2012 by Polish Neuroscience Society - PTBUN, Nencki Institute of Experimental Biology
Evidence suggests that children with autism spec-
trum disorder (ASD) have a greater susceptibility to
heavy-metal intoxication than typically developing
children (Holmes et al. 2003, Kern and Jones 2006,
Rose et al. 2008, Nataf et al. 2008, James et al. 2009,
Geier et al. 2009a, Majewska et al. 2010, Youn et al.
2010, Kern et al. 2011a). For example, children with
ASD have been found to have low plasma glutathione
(GSH) and sulfate (SO4) levels (Waring and Klovrza
2000, James et al. 2004, 2006, 2009, Geier and Geier
2006, Geier et al. 2009c, Pasca et al. 2009, Adams et
al. 2011), both of which are critically important for
detoxification (Gutman 2002, Kern et al. 2004).
Expressions such as “poor detoxifiers” and “poor
excretors” have been used in reference to those with
ASD (Holmes et al. 2003). In a recent analysis, DeSoto
and Hitlan (2010) found that there are 58 research
articles which provide empirical evidence relevant to
the question of a link between autism and one or more
heavy metals. Of those 58 articles, 43 supported a sta-
tistically significant link between autism and exposure
to toxic metals while 15 showed no statistically sig-
nificant evidence of a link between metals and autism.
Thus, 74% of the studies examined showed a signifi-
cant relationship between ASD and toxic metals.
Moreover, several recent studies have shown that the
greater the toxic metal body burden in a child, the
worse the autism symptoms that the child experiences
Evidence of parallels between mercury intoxication
and the brain pathology in autism
Janet K. Kern1,2*, david A. Geier1,3, tapan Audhya4, Paul G. King3, Lisa K. Sykes3, and Mark r. Geier5
1Institute of Chronic Illnesses, Inc., Silver Spring, Maryland, USA; 2University of Texas Southwestern Medical Center at
Dallas, Dallas, Texas, USA , *Email: firstname.lastname@example.org; 3CoMeD, Inc., Silver Spring, Maryland, USA; 4Vitamin
Diagnostics, Cliffwood Beach, New Jersey, USA; 5ASD Centers, LLC, Silver Spring, Maryland, USA
The purpose of this review is to examine the parallels between the effects mercury intoxication on the brain and the brain
pathology found in autism spectrum disorder (ASD). This review finds evidence of many parallels between the two,
including: (1) microtubule degeneration, specifically large, long-range axon degeneration with subsequent abortive axonal
sprouting (short, thin axons); (2) dentritic overgrowth; (3) neuroinflammation; (4) microglial/astrocytic activation; (5) brain
immune response activation; (6) elevated glial fibrillary acidic protein; (7) oxidative stress and lipid peroxidation; (8)
decreased reduced glutathione levels and elevated oxidized glutathione; (9) mitochondrial dysfunction; (10) disruption in
calcium homeostasis and signaling; (11) inhibition of glutamic acid decarboxylase (GAD) activity; (12) disruption of
GABAergic and glutamatergic homeostasis; (13) inhibition of IGF-1 and methionine synthase activity; (14) impairment in
methylation; (15) vascular endothelial cell dysfunction and pathological changes of the blood vessels; (16) decreased
cerebral/cerebellar blood flow; (17) increased amyloid precursor protein; (18) loss of granule and Purkinje neurons in the
cerebellum; (19) increased pro-inflammatory cytokine levels in the brain (TNF-α, IFN-γ, IL-1β, IL-8); and (20) aberrant
nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). This review also discusses the ability of mercury to
potentiate and work synergistically with other toxins and pathogens in a way that may contribute to the brain pathology in
ASD. The evidence suggests that mercury may be either causal or contributory in the brain pathology in ASD, possibly
working synergistically with other toxic compounds or pathogens to produce the brain pathology observed in those diagnosed
with an ASD.
Key words: autism, autism spectrum disorder (ASD), mercury (Hg), toxicity, brain pathology
Correspondence should be addressed to J.K. Kern
Received 03 August 2011, accepted 21 May 2012
114 J.K. Kern et al.
(Holmes et al. 2003, Nataf et al. 2006, Geier and Geier
2007, Geier et al. 2009b, Adams et al. 2009, Kern et al.
2010, Elsheshtawy et al. 2011, Lakshmi Priya and
Geetha 2011). Although studies have shown an asso-
ciation between autism and various toxic metals, such
as cadmium, lead, and arsenic, the bulk of the research
focused on mercury (Hg).
Mercury has a plethora of negative effects on the
brain that are comprehensive and wide-ranging. In
mercury intoxication, multiple systems are targeted.
There does not appear to be one single target effect,
but numerous consequences and cascades of events in
the brain following mercury exposure. If mercury
plays a causal or contributory role in the brain pathol-
ogy of ASD, then the brain pathology seen in mercury
intoxication should be similar to the brain pathology in
ASD. Limited work has been done to examine or com-
pile the similarities between the effects of mercury
intoxication in brain and the pathology found in the
brains of those with ASD. In 2000, Bernard and coau-
thors documented the similarities between the symp-
toms in autism and mercury exposure, and described
brain changes relevant to both mercury and autism.
However, since that time, an ever-increasing body of
evidence elucidating the specific neurological effects
of mercury on the brain has come to light.
This review of the recent literature reveals impor-
tant parallels between the effects of Hg intoxication on
the brain and the brain pathology found in ASD. Each
section will explain the type of pathology evident from
mercury intoxication and then the brain pathology
associated with ASD. Each section will end with a
summary statement. The discussion begins with mer-
cury-induced morphological changes to the neuron.
MIcrotuBuLE And nEurItE
Evidence of microtubule and neurite
degeneration from mercury exposure
Mercury can cause neuronal axons to degenerate
because mercury disrupts the structure of the axon or
neurite, causing it to break apart and depolymerize
(Choi et al. 1981, Vogel et al. 1985, Leong et al. 2001,
Castoldi et al. 2003). A critical structural component of
the neurite membrane is tubulin, a globular protein
(Leong et al. 2001). Under normal conditions, tubulin
molecules link together (end to end) to form microtu-
bules which provide the structure or scaffolding
required by axons and dendrites (Yu et al. 2000, Leong
et al. 2001). Guanosine triphosphate (GTP) continually
binds to the tubulin and provides the energy that
allows the tubulin proteins to remain linked together.
However, when mercury is present in the brain, mer-
cury binds to the GTP binding site of the beta subunit
of the linked tubulin proteins, displacing the GTP.
Because bound GTP provides the energy that allows
the tubulin proteins to link and to remain linked
together, the presence of mercury at the GTP binding
sites stops the supporting energy transfer, which
breaks the links between the tubulin subunits and dis-
rupts this scaffolding. As a consequence, the microtu-
bules break apart and the axons and neurites collapse
or degenerate. This degeneration is also referred to as
process retraction (Choi et al. 1981, Leong et al. 2001,
Castoldi et al. 2003). The progressive degeneration is
presumably mediated through mercury binding to free
sulfhydryl groups both on the ends and on the surface
of the microtubules (Castoldi et al. 2003). Mercury has
a strong general affinity for, and binds with, sulfhy-
dryl (-SH) groups. Moreover, mercury also has a high
affinity for the sulfhydryls in the cytoskeletal proteins
in neurons (Castoldi et al. 2003, Stoiber et al. 2004,
Aschner et al. 2010). Furthermore, the effect that mer-
cury has on microtubules and the subsequent axonal
degeneration is unique to mercury. Other toxic metals,
e.g. lead, manganese, cadmium, aluminum, do not
show this effect (Leong et al. 2001).
This mercury-induced degeneration of the neurite
caused by the binding of mercury to the tubulin has
been shown in both in vitro and in vivo studies by sev-
eral researchers. Leong and colleagues (2001), for
example, applied a metal chloride solution (2 μl) of Hg
(10-7 M) directly onto individual growth cones and
found disruption of their membrane structure and lin-
ear growth rates in 77% of all nerve growth cones,
disintegrated tubulin/microtubule structure, and neu-
ronal somata sprouting failure. In other words, the
axon degenerated. Vogel and coauthors (1985) docu-
mented that depolymerization (axon degeneration)
occurred at concentrations above 1.0 × 10-5 M methyl-
mercury (MeHg). MeHg was bound to free sulfhydryl
groups exposed on the surface and at the ends of
microtubules. Pendergrass and colleagues (1997)
exposed rats to Hg0 at concentrations present in the
‘mouth air’ of some humans with many amalgam fill-
ings and found that by day 14 of exposure, the pres-
Mercury and the brain pathology in autism 115
ence of tubulin was decreased by 41–74%. Pamphlett
and Png (1998) looked for signs of damage to the motor
and sensory neurons of mice that had been exposed to
inorganic mercury and found that mercury “shrinks
motor axons.” The authors found that, after thirty
weeks of exposure to either 1 or 2 μg/g of mercuric
chloride, fewer large myelinated axons were seen in
the Hg-injected groups than in the controls. They also
found a slight increase in numbers of small axons in
the posterior roots of mice exposed to 1 μg/g of Hg.
Importantly, the loss of large myelinated axons or
the selective vulnerability of large axons reported in
the Pamphlett and Png (1998) study has been shown by
others. Stankovic and coauthors (2005), for example,
examined the effects of Hg on motor neurons and
found axonal degeneration, atrophy, and hypertrophy
of axons, with large caliber axons being selectively
vulnerable to the Hg. Another example is from a study
by Stankovic (2006), who found atrophy principally to
large myelinated fibers, a subpopulation of axons.
Again, Mitchell and Gallagher (1980) had previously
found methyl mercuric acetate (MeHgOAc) caused
axonal degeneration in large myelinated fibers. It is
important to note that projection or long-range neurons
have, in general, bigger cell bodies and axons than
local circuit (LC) neurons (Jacquin et al. 1989, Taylor
Neurites (axons and dendrites) provide the connec-
tions between neurons, and the connections between
neurons form the neural circuitry of the brain. The
connectivity of the neural circuitry allows for interac-
tion within a brain region and between distinct brain
regions. Retraction of processes or loss of these con-
nective axons, as described above, leads to loss of con-
nectivity in the brain. The following section examines
the evidence for process retraction and abnormal con-
nectivity found in autism.
Evidence of neurite degeneration/process
retraction (loss of axons) and loss of connectivity
Recent studies have shown evidence of process
retraction or loss of axons in autism. Morgan and col-
leagues (2010), for example, examined the dorsolateral
prefrontal cortex of male cases with autism (n=13) and
control cases (n=9), and found process retraction and
thickening in the males with autism but not in the con-
trol males. Zikopoulos and Barbas (2010) examined
changes in axons in postmortem human brain tissue
below the anterior cingulate cortex (ACC), orbitofron-
tal cortex (OFC), and lateral prefrontal cortex (LPFC)
in autism and found a decrease in the largest axons that
communicate over long distances and an excessive
number of thin axons that link neighboring areas.
Numerous studies have reported abnormal connec-
tivity in those with ASD (Wan and Schlaug 2010).
Several terms are used to describe this impaired neu-
ron connectivity found in those with autism, such as
“underconnectivity”, “impaired connectivity”, “dis-
rupted connectivity”, or “altered brain connectivity”
(Belmonte et al. 2004, Wan and Schlaug 2010, Wass
2011). More specifically, studies have reported under-
connectivity in long-range connections and overcon-
nectivity in short-range or local networks, with the
frontal and temporal lobes being the most affected
(Wass 2011). Examples of studies that found abnormal
connectivity (both long-range underconnectivity and
short-range overconnectivity) in those with autism are
Barttfeld and coauthors (2011) used electroencepha-
lography (EEG) to assess dynamic brain connectivity
in ASD focusing in the low-frequency (delta) range
and found that those with ASD lacked long-range con-
nections and had increased short-range connections.
Interestingly, as ASD severity increased, short-range
coherence was more pronounced and long-range coher-
ence decreased. Using magnetoencephalographic
(MEG), Pollonini and colleagues (2010) analyzed
brain connectivity based on Granger causality com-
puted from activity in eight subjects with autism and
eight normal individuals. They found measurable con-
nectivity differences between the two groups.
The cortical underconnectivity theory in autism was
investigated by examining the neural bases of the visu-
ospatial processing in one study in high-functioning
autism. Using a combination of behavioral, functional
magnetic resonance imaging, functional connectivity,
and corpus callosum morphometric methodological
tools, Damarla and coauthors (2010) found that the
autism group had lower functional connectivity
between the higher-order working memory/executive
areas and the visuospatial regions (between frontal and
Several other studies using various methods, such as
magnetic resonance imaging (MRI) and white matter
parcellation technique, have examined connectivity in
autism. These studies have all shown that functional
116 J.K. Kern et al.
connectivity among regions of autistic brains is dimin-
ished (Herbert et al. 2004, 2005, Herbert 2005). For
example, Ebisch and colleagues (2011), using func-
tional magnetic resonance imaging (fMRI), found
reduced functional connectivity in ASD, compared
with controls, between anterior and posterior insula
and specific brain regions involved in emotional and
sensory processing. They stated that the functional
abnormalities in a network involved in emotional and
interoceptive awareness might be at the basis of altered
emotional experiences and impaired social abilities in
For local, or short-range, overconnectivity, several
studies have suggested regional brain overgrowth in
autism. Stiglerand coauthors (2011) conducted a review
of relevant structural and functional MRI studies in
ASDs and reported “early rapid brain overgrowth in
affected individuals”. Similarly, from their imaging
work, Courchesne and colleagues (2003) reported evi-
dence of brain overgrowth in the first year of life in
those with an autism diagnosis. They also reported that
the excessive growth is followed by abnormally slow
or arrested growth (Courchesne 2004). Again, Santos
and coauthors (2010) found that the overgrowth clearly
begins before 2 years of age. They conducted postmor-
tem analysis of the brains of four young patients with
autism and three controls of comparable age and found
neuronal overgrowth in those with autism. Schumann
and coauthors (2010) conducted a structural magnetic
resonance imaging (MRI) longitudinal study of brain
growth in toddlers at the time symptoms of autism are
becoming clinically apparent and at multiple times
thereafter (1.5 years up to 5 years of age), and found
both cerebral gray and white matter were significantly
enlarged in toddlers with ASD, with the most severe
enlargement occurring in frontal, temporal, and cingu-
late cortices. The amygdale has also shown over-
growth. Measuring amygdala volumes on magnetic
resonance imaging scans from 89 toddlers who were
1–5 years of age (mean = 3 years), Schumann and col-
leagues (2009) found overgrowth beginning before 3
years of age. Similar to the Barttfeld and coauthors
(2011) study mentioned earlier, the extent of abnormal
connectivity was associated with the severity of clini-
There are many more studies that suggest problems
with connectivity in those with an ASD diagnosis. For
a complete review of connectivity and list of the cur-
rent studies that suggest abnormal brain connectivity
in ASD, please see Wass (2011). As Wass (2011)
observes in this recent review of connectivity in ASD,
there is “considerable convergent evidence suggesting
that connectivity is disrupted in ASD.” From his
review of the literature, he states that the evidence
indicates both local over-connectivity and long-dis-
tance under-connectivity, and that disruptions appear
more severe in the later-developing cortical regions.
Long-range underconnectivity between regions and
short-range over-connectivity appears to be pervasive
in those with autism (Wass 2011). How Hg could cause
long-range underconnectivity between regions and
short-range over-connectivity in autism is discussed
further in following section.
How Hg exposure could result in both the long-
distance under-connectivity and local over-
connectivity seen in autism
To date, there are only theories as to the cause of the
short-range overconnectivity in ASD (Courchesne et
al. 2011). This section will discuss how short-range
overconnectivity could result from the loss of long-
As mentioned earlier, mercury seems to preferen-
tially target large axons and to cause retraction/degen-
eration of those axons (Mitchell and Gallagher 1980,
Stankovic 2006), and the evidence in autism shows
axonal retraction, a decrease in the proportion of larg-
est axons – the ones that communicate over long dis-
tances (Zikopoulos and Barbas 2010, Wass 2011).
Since Hg causes the loss of long-range connectivity
from degeneration of large, long-range axons, it is con-
ceivable that the local outgrowth of axonal sprouting
and dendritic overgrowth is a compensatory mecha-
nism. The following evidence explains this model.
Following traumatic injury to central nervous sys-
tem (CNS) axons, axons undergo what is called regen-
erative sprouting. Regenerative sprouting is when an
injured neuron attempts to reform an injured axon.
However, it is usually referred to as “abortive sprout-
ing” (Schwartz and Flanders 2006), because of the
inability of injured axons to cross the lesion site, to
elongate, and to undergo true axonal regeneration
(Meyer et al. 2009). The shorter the distance between
the regeneration site and its distal target, the more suc-
cessful regeneration of a nerve is likely to be, because
postnatal, mature neuronal axons will only regenerate
for very short distances in the CNS (Fawcett 1992).
Mercury and the brain pathology in autism 117
Although many CNS neurons can survive for years
after injury, the injured axons fail to regenerate beyond
the lesion site in children and adults (Glenn and
Zhigang 2006). The lack of a regenerative response is
due, in part, to the presence of inhibitory molecules
such as myelin-derived proteins or chondroitin sul-
phate proteoglycans (Hill et al. 2001, Seira et al. 2010).
In addition, glial cells in the CNS (both oligodendro-
cytes and astrocytes) at the site of injury produce
inhibitory molecules that inhibit axonal regrowth
(Fawcett 1997, Stichel and Muller 1998, Goldshmit et
al. 2004). As mentioned earlier, the loss of long-range
axons from Hg appears to result in a slight increase in
numbers of small axons (Pamphlett and Png 1998).
Although axonal regeneration is limited, the den-
dritic response to neuronal or axonal injury is over-
growth, i.e. an overproduction of dendritic branches
(Jones and Schallert 1992, Jones 1999). It has been
shown that following damage to connected brain
regions, the brain undergoes an adaptive response
which includes reactive axonal sprouting and an over-
production of dendrites (Jones 1999). Moreover, even
though the overgrowth of dendrites eventually under-
goes pruning, the overgrowth remains increased rela-
tive to controls (Jones and Schallert 1994, Jones 1999).
Several other studies show that dentritic overgrowth
secondary to neuronal injury is followed only by a
partial reduction in the dendritic branching (Jones and
Schallert 1992, Kozlowski et al. 1996, Brown and
Interestingly, in autism specifically, Zikopoulos and
Barbas (2010) found a higher density of small axons
and a significantly higher percentage of axons with
branches compared to control cases, and that most
points of bifurcation were unmyelinated or arose after
thinning of the myelin. In patients with Minamata dis-
ease, caused from methylmercury poisoning (where
the putative source of mercury compound was methyl-
mercury cysteine – MeHgCys– from fish), regenerated
axons were extremely small in size following regen-
erative sprouting and many fibers were found to be
unmyelinated and poorly myelinated (Takeuchi et al.
Section summary statement
Mercury exposure can result in loss of long-range
axons andlong-range underconnectivity and compen-
satory dendritic and axonal sprouting/short-range over-
growth. The deficit of long-range connectivity and
short-range overconnectivity is what is found in the
brain of those with ASD.
The evidence in this section suggests neuronal
injury. Neuronal injury in the CNS would result in
microglial activation. The next section discusses the
evidence for microglia activation and neuroinflamma-
tion in the brain.
MIcroGLIA ActIVAtIon And
Evidence of microglia activation,
neuroinflammation, gliosis, and immune
response in the cnS from Hg exposure
The brain responds to injury by rapidly activating
the brain’s own immune system, largely composed of
glial cells (Streit et al. 2004, Streit and Xue 2009).
Reactive gliosis specifically refers to the accumulation
of enlarged glial cells (microglia and astrocytes)
appearing immediately after a CNS injury has occurred
(Vajda 2002). The presence of gliosis is suggestive of
brain insult and neuroinflammation (Vajda 2002).
Microglia are the smallest of the glial cells and con-
stitute approximately 20% of the glial cell population.
Microglia are the resident macrophages of the central
nervous system (CNS). They are considered to be the
main form of immune defense in the CNS and are
important for maintaining homeostasis. As key cellu-
lar mediators of the neuroinflammatory processes,
microglia are associated with the pathogenesis of
many neurodegenerative and brain inflammatory dis-
eases (Ginhoux et al. 2010), and are involved in acute
and chronic neuroinflammation (Streit et al. 2004,
Streit and Xue 2009).
Once activated, microglia release nitric oxide (NO)
and superoxide as a cytotoxic attack mechanism
(Colton and Gilbert 1993). Reactive oxygen and nitro-
gen species (ROS and RNS) derived from NO and
superoxide may also cause local cellular damage by
reacting with proteins, lipids and nucleic acids (Valko
et al. 2007). In addition, production of NO following
microglial activation causes a decline in cellular glu-
tathione (GSH) levels, leading to brain oxidative
damage (Moss and Bates 2001). According to Stichel
and Muller (1998), astrocytes in the adult show a vig-
orous response to injury; they become hypertrophic,
proliferative as they upregulate expression of glial
118 J.K. Kern et al.
fibrillary acidic protein (GFAP), and form a dense
network of glial processes both at and extending from
the lesion site. Streit and coauthors (2004) state that in
the case of chronic neuroinflammation, the cumula-
tive ill effects of microglial and astrocytic activation
can contribute to and expand the initial neurodestruc-
tion, thus maintaining and worsening the disease
process through their actions. Evidence suggests that
the collateral neural damage can involve loss of syn-
aptic connections in the brain (Gehrmann et al.
Numerous studies have shown that mercury expo-
sure causes microglial activation, gliosis, neuroin-
flammation, and immune response in the CNS (Castoldi
et al. 2003, Zhang et al. 2011). Specific examples of
these processes include:
Using cell cultures of different complexity, isolated
microglia were found to be directly activated by non-
cytotoxic MeHgCl treatment by Eskes and colleagues
(2002). The authors stated that microglial cells react
just after a neurotoxic insult. Moreover, the interaction
between activated microglia and astrocytes can
increase local IL-6 release, which may cause astrocyte
reactivity and neuroprotection.
In mice, Fujimura and coauthors (2009) adminis-
trated 30 ppm of methylmercury (MeHg) in drinking
water for 8 weeks. They found a decrease in the num-
ber of neurons, an increase in the number of migratory
astrocytes and microglia/macrophages, and necrosis
and apoptosis in the cerebral cortex. In rats, Monnet-
Tschudi and colleagues (1996) found a microglial
response to long-term exposure to subclinical doses of
Hg. These two studies suggest that the dose of Hg
does not have to be high to get microglial activation.
Again in rats, Gajkowska and colleagues (1992)
administered a single dose (6 mg/kg body weight) of
HgCl2 to rats and found (after 18 hours) accumulation
of dense deposits of mercury in nerve and glial cell
cytoplasm with an increase in the quantity of micro-
glia in the experimental group. Roda and coauthors
(2008) investigated the effects of perinatal (GD7-
PD21) administration of MeHg in drinking water (0.5
mg/kg bw/day) on cerebellum of immature (PD21)
and mature (PD36) rats. They found reactive gliosis,
e.g. a significant increase in Bergmann glia of the ML
and astrocytes of the IGL, identified by their expres-
sion of glial fibrillary acidic protein. Vicente and
coauthors (2004) also found glial involvement in the
MeHg-induced neurotoxicity in rats.
Similarly, in monkeys, Charleston and colleagues
(1996) examined effects of long-term subclinical expo-
sure to methylmercury on microglia in the thalamus of
the Macaca fascicularis and found microglia showed a
significant increase in the 18-month and clearance
exposure groups. Earlier, Charleston and coauthors
(1995) examined effects of long-term subclinical expo-
sure to methylmercury and mercuric chloride [HgCl2,
which directly releases Hg2+(the putative inorganic mer-
cury – IHg)] and found that the astrocytes and microglia
in the MeHg exposure groups contained the largest
deposits of IHg. They stated that all neurons in the
18-month exposure group contained deposits of IHg;
however, these total deposits were considerably smaller
than those within the astrocytes and microglia.
In humans, Eto and colleagues (1999) found changes
produced by organic mercury in the brain of patients
with Minamata disease who had acute onset of symp-
toms, and those who died within 2 months; they
showed loss of neurons with reactive proliferation of
glial cells. Interestingly, histochemistry of the mercury
revealed that inorganic mercury was present in the
Numerous studies have also shown that mercury
exposure results in an increase in glial fibrillary acidic
protein (GFAP). GFAP is elevated in acute and chronic
situations of nerve cell damage and a marker of astro-
glial activation (Ahlsen et al. 1993). Examples are as
El-Fawal and coauthors (1996) examined serum
autoantibodies (Ig) to neurotypic and gliotypic pro-
teins, myelin basic protein (MBP) and glial fibrillary
acid protein (GFAP) as markers of subclinical neuro-
toxicity from methyl mercury (MeHg). They found
that MeHg resulted in increased GFAP in the cerebel-
lum at 14 days and elevation of several of the autoanti-
bodies tested. Using GFAP as a quantitative marker of
neuronal injuries on the central nervous system,
Toimela and Tähti (1995) found that staining with
monoclonal antibody showed GFAP induction after
In a five laboratory collaborative study, Elsner and
colleagues (1988) evaluated the effects of methylmer-
cury on the in utero rat pups by treating rat dams at
days 6 to 9 of gestation. They examined behavioral
outcomes and GFAP and S-100 protein concentration
in the rat pups, and found dose-dependent effects with
increased GFAP concentration in the cerebellar ver-
mis, increased auditory startle amplitude, and other
Mercury and the brain pathology in autism 119
behavioral outcomes. Moreover, the study showed
comparable sensitivities for the behavioral testing bat-
tery and the neurochemical assays.
Numerous studies show the activation of microglia
and signs of neuroinflammation, gliosis, and immune
response from mercury exposure in the brain of mam-
mals. The evidence for the same findings in autism is
the topic of the next section.
Evidence of microglial activation,
neuroinflammation, gliosis, and immune
response in autism
Evidence suggests that children with autism suffer
from an ongoing inflammatory process in different
regions of the brain involving microglial activation
(Enstrom et al. 2005, Vargas et al. 2005, Zimmerman
et al. 2005, Morgan et al. 2010). Herbert (2005) pointed
out that the autistic brain is not simply wired differ-
ently, but that neuroinflammation is a part of the
pathology in autism.
Vargas and coauthors (2005), for example, exam-
ined brain tissue and cerebral spinal fluid (CSF) in
those with autism. For the morphological studies, brain
tissues from the cerebellum, midfrontal, and cingulate
gyrus were obtained at autopsy from 11 patients with
autism. Fresh-frozen tissues from seven patients and
CSF from six living patients with autism were used for
cytokine protein profiling. The authors found active
neuroinflammatory process in the cerebral cortex,
white matter, and notably in cerebellum of patients
with autism, with marked activation of microglia and
astroglia. The authors stated that the CSF showed a
unique proinflammatory profile of cytokines. The
authors stated that the pattern of cellular and protein
findings suggests the brain’s own immune system (not
immune abnormalities from outside the brain) and that
the neuroinflammatory process appears to be an ongo-
ing and chronic mechanism of CNS dysfunction.
Morgan and colleagues (2010) examined the dorso-
lateral prefrontal cortex of male cases with autism
(n=13) and control cases (n=9) and found microglial
activation and increased microglial density in the dor-
solateral prefrontal cortex in those with autism. They
also noted process retraction and thickening, and
extension of filopodia (small protrusions sent out from
a migrating cell in the direction that it wants to move)
from the processes. The authors stated that the micro-
glia were markedly activated in 5 of 13 cases with
autism, including 2 of 3 under age 6, and marginally
activated in an additional 4 of 13 cases. The authors
stated that because of its early presence, microglial
activation may play a central role in the brain patho-
genesis of autism.
Several studies have shown that GFAP levels are
increased in autism. An autopsy report by Bailey and
coauthors (1998), found that the Purkinje cell loss was
sometimes accompanied by gliosis and an increase in
GFAP. Laurence and Fatemi (2005) examined levels of
GFAP in the frontal, parietal, and cerebellar cortices
using age-matched autistic and control postmortem
specimens. GFAP was significantly elevated in all
three brain areas. The authors stated that the elevated
GFAP confirms microglial and astroglial activation in
autism and indicates gliosis, reactive injury, and per-
turbed neuronal migration processes. A study by
Ahlsen and colleagues (1993) examined the levels of
GFAP in the CSF of children with autism, and found
their average GFAP was three times higher than it was
in the control group. The authors stated that the results
could implicate gliosis and unspecified brain damage
in children with autism. Likewise, Rosengren and col-
leagues (1992) found GFAP levels in CSF in children
with autism were higher than those in normal control
children of the same age range. The authors stated that
the high levels of GFAP in combination with normal
S-100 protein concentrations in CSF indicate reactive
astrogliosis in the CNS.
Fatemi and coauthors (2008) investigated whether
two astrocytic markers, aquaporin 4 and connexin 43,
are altered in Brodmann’s Area 40 (BA40, parietal
cortex), Brodmann’s Area 9 (BA9, superior frontal
cortex), and the cerebella of brains of subjects with
autism and matched controls. The authors reported
that the findings demonstrated significant changes in
two astrocytic markers in the brains from subjects
Section summary statement
Mercury exposure can result in activation of the
brain’s immune system characterized by elevated
microglial cells and astrocytes, which is also found
in the brains of those with ASD. Although this reac-
tion is unspecific and may be triggered by many
factors, the lack of microglial activation in either
mercury intoxication or autism would decrease the
probability of such causal connection.
120 J.K. Kern et al.
oXIdAtIVE StrESS And LIPId
Free radicals and other reactive oxygen species
(ROS) are produced in all species. Any free radical
involving oxygen can be referred to as ROS, e.g. nitric
oxide (NO). Free radicals and other ROS are unstable
atoms, molecules, or ions with unpaired electrons.
They are harmful because the unpaired electron oxida-
tively reacts with other ions and molecules, or they
“steal” an electron from other molecules to pair that
electron. This produces disruption to other molecules
and damage to cells (Gutman 2002). One of the main
problems is that ROS “steal” electrons from lipid
membranes (the cell membrane of most living organ-
isms is made of a lipid bilayer). The oxidative degrada-
tion of the lipid membrane is referred to as lipid per-
oxidation. Lipid peroxidation results in loss of mem-
brane integrity and fluidity, which ultimately leads to
cell death (Esterbauer et al. 1991, Efe et al. 1999). ROS
also react with proteins and nucleic acids which can
lead to cell death via apoptosis or necrosis (Kannan
and Jain 2000).
Under normal conditions, a dynamic equilibrium
exists between the production of ROS and the antioxi-
dant capacity of the cell (Stohs 1995, Granot and
Kohen 2004). Normally, the ROS within the cells are
neutralized by antioxidant defense mechanisms.
Superoxide dismutase (SOD), catalase, and glutathione
peroxidase (GPx) are the primary enzymes involved in
direct elimination of ROS, whereas glutathione
reductase and glucose-6-phosphate dehydrogenase are
secondary antioxidant enzymes, which help in main-
taining a steady concentration of reduced glutathione
(GSH) and NADPH necessary for optimal functioning
of the primary antioxidant enzymes (Chance 1954,
Maddipati and Marnett 1987, Vendemiale et al. 1999).
GSH is the most important antioxidant for detoxifica-
tion and is important for the elimination of environ-
mental toxins. Oxidative stress occurs when there is an
imbalance between free radicals and the ability to neu-
tralize them (i.e., an excess of pro-oxidants, a decrease
in antioxidant levels, or both).
The brain is highly vulnerable to oxidative stress
due to its limited antioxidant capacity, higher energy
requirement, and higher amounts of lipids and iron
(Juurlink and Paterson 1998). The brain makes up
about 2% of body mass but consumes 20% of meta-
bolic oxygen. Neurons use the vast majority of the
body’s energy (Shulman et al. 2004). Because neurons
lack the capacity to produce GSH, the brain has a lim-
ited capacity to detoxify ROS. Therefore, neurons are
the first cells to be affected by the increase in ROS
and/or a shortage of antioxidants. As a result, they are
susceptible to oxidative stress. Antioxidants are
required for neuronal survival during the early critical
developmental period (Perry et al. 2004). Children are
more vulnerable than adults are to oxidative stress
because of their naturally lower GSH levels from con-
ception through infancy (Ono et al. 2001, Erden-Inal et
al. 2002). The risk created by this natural deficit in
detoxification capacity in infants is increased by the
fact that some environmental factors that induce oxi-
dative stress are found at higher concentrations in
developing infants than in their mothers, and these
preferentially accumulate in the placenta and the
developing fetus. Taken together, these studies suggest
that the developing brain is highly vulnerable to oxida-
Evidence that Hg induces oxidative stress, lipid
peroxidation, and altered glutathione levels and
activity in the cnS
Numerous studies show that Hg exposure induces
oxidative stress and lipid peroxidation in the CNS, as
well as has a negative impact on glutathione and thiols
(Ueha-Ishibashi et al. 2004, Huang et al. 2008, Monroe
and Halvorsen 2009, Hoffman et al. 2011). Some rele-
vant examples of pertinent studies include:
Huang and coauthors (2008) studied low-dose and
long-term exposure of methylmercury (MeHg) in
mice. They found significant Hg accumulation and
biochemical alterations in brain regions and/or other
tissues, including the increase of lipid peroxidation
(LPO) production, influence of Na+/K+-ATPase activi-
ties and nitric oxide (NO) levels. Again in 2011, Huang
and his colleagues examined the underlying mecha-
nisms of neurotoxic effects of both methylmercury
(MeHg) and mercury chloride (HgCl2) in mice and
found that the alteration of lipid peroxidation (LPO),
Na+/K+-ATPase activities, and nitric oxide (NOx) in the
brain tissues contributed to the observed neurobehav-
ioral dysfunction and hearing impairment (Huang et
Even studies that gave antioxidants in conjunction
with mercury found oxidative stress in the brain.
Glaser and coauthors (2010), for example, gave adult
Mercury and the brain pathology in autism 121
male mice MeHg orally in drinking water (40 mg/ L-1),
and simultaneously administrated daily subcutaneous
injections of sodium selenite (Na2SeO3). Although
there was a reduction in cells with metal deposition in
the brain, there was still an increase in lipid peroxida-
tion in the brain.
Many studies that show mercury induces oxidative
stress and lipid peroxidation in the brain also find a
concomitant decrease in glutathione levels, as well as
alterations in GSH-related enzymes (Ou et al. 1999,
Manfroi et al. 2004, Franco et al. 2006, 2007, 2009,
2010, Stringari et al. 2006, 2008, Yin et al. 2007,
Aschner et al. 2007). Franco and colleagues (2010), for
example, found that incubation of mouse brain mito-
chondria with MeHg induced a significant decrease in
mitochondrial function, which was correlated with
decreased GSH levels and increased generation of
ROS and lipid peroxidation.
As reported by Stringari and coauthors (2006), and
other studies (Manfroi et al. 2004, Franco et al. 2006),
the GSH antioxidant system is a significant molecular
target of MeHg and during the early postnatal period,
mercury exposure results in decreased GSH levels and
decreased activities of GSH-related enzymes.
Moreover, Stringari and colleagues (2008) found, in a
follow up study, that mercury exposure effectively
inhibited the developmental profile of the cerebral
GSH antioxidant system during the early postnatal
period. The authors went on to state that the inhibition
of the maturation the GSH antioxidant system might
contribute to the oxidative damage seen after prenatal
MeHg exposure, because even though the cerebral
mercury concentration decreased later postnatally, the
GSH levels, glutathione peroxidase (GPx) and glutathi-
one reductase (GR) activities remained decreased in
MeHg-exposed mice. According to Stringari and col-
leagues (2008), the evidence corroborates previous
reports that indicate prenatal exposure to MeHg affects
the GSH antioxidant systems by inducing biochemical
alterations that persist even when mercury tissue levels
decreased to the same levels as those in the controls.
This early exposure induces pro-oxidative damage and
permanent functional deficits in the developing CNS.
Ueha-Ishibashi and coauthors (2004) examined the
effects of Thimerosal (sodium ethylmercury thiosali-
cylate, Na+ EtHgSal–), an organomercurial preservative
in vaccines, on cerebellar neurons dissociated from
2-week-old rats, as compared to methylmercury, and
found that both agents (at 1 μM or more) similarly
decreased the cellular content of glutathione in a con-
centration-dependent manner, suggesting an increase
in oxidative stress. As evident in this study, it is impor-
tant to note that many of the studies mentioned in this
section show a dose-dependent affect, i.e., the greater
the levels of Hg, the higher the levels of oxidative
Evidence of oxidative stress, lipid peroxidation,
and altered glutathione levels and activity in the
brain in autism
Three postmortem studies published in 2008 revealed
that affected areas of the brain in children with autism
showed accelerated cell death under conditions of oxi-
dative stress (Lopez-Hurtado and Prieto 2008, Evans
et al. 2008, Sajdel-Sulkowska et al. 2008). Evans and
coauthors (2008), for example, evaluated the oxidative
stress metabolites of carboxyethyl pyrrole (CEP) and
isolevuglandin (isoLGE2-protein adducts in corti-
cal brain tissues of subjects diagnosed with autism.
Significant immunoreactivity toward these markers of
oxidative damage in the white matter, often extending
well into the grey matter of axons, was found in every
case of autism examined. These investigators reported
that the striking thread-like pattern appears to be a
hallmark in the brains of those diagnosed with ASD,
as it was not seen in any control brains, young or aged,
used as controls for the oxidative assays. In another
study, the density of lipofuscin, a matrix of oxidized
lipid and cross-linked protein that forms as a result of
oxidative injury in the tissues, was observed to be
greater in cortical brain areas concerned with commu-
nication in subjects diagnosed with autism (Lopez-
Hurtado and Prieto 2008) than in controls. Lipofuscin
was previously demonstrated to be a depot for mercury
in human brain autopsy specimens from mercury-in-
toxicated patients (Opitz et al. 1996). Finally, and per-
haps most importantly, Sajdel-Sulkowska and col-
leagues (2008) evaluated cerebellar levels of the oxida-
tive stress marker 3-nitrotyrosine (3-NT), mercury,
and the antioxidant selenium in subjects diagnosed
with autism and in control subjects. These researchers
found that there were significant increases in the mean
cerebellar levels of 3-NT and in the ratio of mercury/
selenium in the brains of subjects diagnosed with
autism when compared to controls. Importantly, there
was a significant dose-dependent positive correlation
between the oxidative stress markers and total mercury
122 J.K. Kern et al.
levels. This dose-dependent effect is seen in many
studies (as shown in the previous section) in animals
In 2009, Sajdel-Sulkowska and colleagues also pub-
lished a study where they examined oxidative damage
in the cerebellum of those with ASD by measuring
8-hydroxydeoxyguanosine (8-OH-dG), a marker of
DNA modification, in a subset of cases they also ana-
lyzed for 3-NT. The authors found that cerebellar
8-OH-dG showed an upward trend toward higher lev-
els with an increase of 63.4% observed in those with
autism. Analysis of cerebellar neurotrophin-3 (NT-3)
showed a statistically significant (P=0.034) increase
(40.3%) in those with autism. Furthermore, there was
a significant positive correlation between cerebellar
NT-3 and 3-NT. The authors stated that the altered
levels of brain NT-3 are likely to contribute to autistic
pathology not only by affecting brain axonal targeting
and synapse formation but also by further exacerbat-
ing oxidative stress and possibly contributing to
Purkinje cell abnormalities.
Later in 2011, Sajdel-Sulkowska and coauthors
examined whether the increase in oxidative stress in
ASD is brain region-specific. They compared brain
region-specific NT-3 expression between those with
ASD and control cases. The 3-NT and NT-3 were
measured with specific ELISAs in individual brain
regions of two autistic and age- and postmortem
interval (PMI)-matched control donors. The authors
found that the levels of 3-NT, ranging from 1.6 to
12.0 pmol/g, were uniformly low in all brain regions
examined in controls. However, there was a large
degree of variation in 3-NT levels and its maximum
levels were much higher, ranging from 1.7 to
281.2 pmol/g, among individual brain regions in those
with autism. The brain regions with the increased
3-NT levels and the magnitudes of the increase were
both different in the two autistic cases. In the brain of
the older case, the brain regions with highest levels of
3-NT included the orbitofrontal cortex (214.5 pmol/g),
Wernicke’s area (171.7 pmol/g), cerebellar vermis
(81.2 pmol/g), cerebellar hemisphere (37.2 pmol/g),
and pons (13.6 pmol/g) (brain areas associated with
the speech processing, sensory and motor coordina-
tion, emotional and social behavior, and memory).
Brain regions that showed 3-NT increases in both of
those with ASD included the cerebellar hemispheres
and putamen. Consistent with their earlier report, the
researchers found an increase in NT-3 levels in the
cerebellar hemisphere in both of the brains from sub-
jects who had been diagnosed with ASD. They also
detected an increase in NT-3 level in the dorsolateral
prefrontal cortex (BA46) in the brain from the older
individual and in the Wernicke’s area and cingulate
gyrus in the brain of the younger case.
Many studies have shown that plasma GSH levels
are low and biomarkers of oxidative stress are high in
ASD (Geier et al. 2009c); moreover, in recent study
by Chauhan and coauthors (2012), they found that the
same is true when directly measuring brain tissue in
ASD. They compared DNA oxidation and glutathi-
one redox status in postmortem brain samples from
the cerebellum and frontal, temporal, parietal and
occipital cortex from autistic subjects and age-
matched normal subjects. The authors reported that
DNA oxidation was significantly increased by two-
fold in frontal cortex, temporal cortex, and cerebel-
lum in individuals with autism as compared with
control subjects. Moreover, the levels of reduced glu-
tathione GSH were significantly reduced and the
levels of oxidized glutathione GSSG were signifi-
cantly increased in the cerebellum and temporal cor-
tex in the brain samples from the group with autism
as compared to the corresponding levels in the con-
trol brain samples.
Earlier, Chauhan and colleagues (2011) studied
the levels of mitochondrial electron transport chain
(ETC) complexes in brain tissue samples from the
cerebellum and the frontal, parietal, occipital, and
temporal cortices of subjects with autism and age-
matched control subjects. The subjects were divided
into two groups according to their ages: Group A
(children, ages 4–10 years) and Group B (“adults”,
ages 14–39 years). A significant increase in the lev-
els of lipid hydroperoxides, an oxidative stress
marker, was observed in the cerebellum and tempo-
ral cortex in the children with autism as compared
to the levels in the controls. The authors also found
evidence of mitochondrial dysfunction in the brain,
which will be discussed further in the following
Section summary statement
Mercury intoxication can result in elevated oxida-
tive stress markers and lowered GSH levels in the
brain. Both are present in the brains of persons with
Mercury and the brain pathology in autism 123
Evidence of mitochondrial damage and
dysfunction in the brain from Hg exposure
Numerous studies show that Hg causes systemic
mitochondrial dysfunction (Stohs and Bagchi 1995,
Stacchiotti et al. 2010, Belyaeva et al. 2011) and causes
mitochondrial dysfunction in the brain (Stohs and
Bagchi 1995, Allen et al. 2001, Castoldi et al. 2003,
Limke et al. 2004, Yin et al. 2007, Dreiem and Seegal
2007, Franco et al. 2007, 2010, Monroe and Halvorsen
2009, Kaur et al. 2010, Migdal et al. 2010). Although
mercury, as stated by Yin and coauthors (2007), “initi-
ates multiple additive or synergistic disruptive mecha-
nisms,” the main mechanism of disruption to mito-
chondrial function appears to result from the mercury-
induced production of ROS. As mentioned before,
Franco and colleagues (2010) found that incubation of
mouse brain mitochondria with MeHg induced a sig-
nificant decrease in mitochondrial function, which
was correlated with decreased GSH levels and increased
generation of ROS and lipid peroxidation. Hg depletes
GSH and protein-bound sulfhydryl groups, resulting
in the production of ROS, and as a consequence, lipid
peroxidation, and specifically mitochondrial lipid per-
oxidation occurs (Stohs and Bagchi 1995, Kaur et al.
2010). As mentioned in a previous section, lipid per-
oxidation results in membrane permeability. Yin and
colleagues (2007), for example, found that methylmer-
cury exposure results in a concentration-dependant
reduction in the inner mitochondrial membrane poten-
tial and increased mitochondrial membrane permea-
bility. Further, Migdal and coauthors (2010) found that
Thimerosal, a mercury derivative composed of ethyl
mercury chloride (EtHgCl) and thiosalicylic acid
(TSA), caused mitochondrial membrane depolariza-
tion and changes in mitochondrial membrane permea-
bility. In addition, methylmercury induces the over-
production of hydrogen peroxide (H2O2), which in
turn, inhibits astrocyte glutamate transporters, and
leads to increased glutamate concentrations and gluta-
mate-induced oxidative stress. Thus, both direct
Hg-induced oxidative stress and glutamate-induced
oxidative stress result in mitochondrial dysfunction
(Allen et al. 2001, Franco et al. 2007).
Although several studies have found that Hg induces
mitochondrial dysfunction secondary to the formation
ROS, Dreiem and Seegal (2007) found that even when
mitochondria are exposed to methylmercury chloride
in conjunction with the antioxidant Trolox (6-hydroxy-
2,5,7,8-tetramethylchroman-2-carboxylic acid, a water-
-soluble derivative of vitamin E), which significantly
reduced MeHgCl-induced ROS levels, it failed to
restore mitochondrial function. The authors found that
mercury also increased mitochondrial calcium levels
in striatal synaptosomes, and proposed that the
increased mitochondrial calcium levels also contribute
to the mitochondrial dysfunction. In other words, the
MeHgCl disrupted calcium homeostasis critical for
The following section discusses the evidence for
mitochondrial dysfunction in the brains of those with
Evidence of mitochondrial damage and
dysfunction in the brain in autism
A recent review by Palmieri and Persico (2010),
reports that a substantial percentage of patients with
ASD display peripheral markers of mitochondrial
energy metabolism dysfunction, such as (a) elevated
lactate, pyruvate, and alanine levels in blood, urine
and/or cerebrospinal fluid, (b) serum carnitine defi-
ciency, and/or (c) enhanced oxidative stress. Other
researchers have also reported evidence of systemic
mitochondrial dysfunction (Giulivi et al. 2010).
Moreover, recent evidence from two postmortem stud-
ies of the brains of those with ASD points specifically
toward abnormalities in mitochondrial function in the
brain. Two examples are as follows:
First, Chauhan and colleagues (2011) studied the
levels of mitochondrial electron transport chain (ETC)
complexes, i.e. complexes I, II, III, IV, and V, in brain
tissue samples from the cerebellum and the frontal,
parietal, occipital, and temporal cortices of subjects
with autism and age-matched control subjects. The
subjects were divided into two groups according to
their ages: Group A (children, ages 4–10 years) and
Group B (“adults”, ages 14–39 years). In Group A, they
observed significantly lower levels: of complexes III
and V in the cerebellum (P<0.05), of complex I in the
frontal cortex (P<0.05), and of complexes II (P<0.01),
III (P<0.01), and V (P<0.05) in the temporal cortex of
children with autism as compared to age-matched con-
trol subjects. In the cerebellum and temporal cortex,
no overlap was observed in the levels of these ETC
complexes between subjects with autism and the con-
124 J.K. Kern et al.
trol subjects. In the frontal cortex of Group A, a lower
level of ETC complexes was observed in a subset of
autism cases, i.e. 60% (3/5) for complexes I, II, and V,
and 40% (2/5) for complexes III and IV. A striking
observation, made by the authors, was that the levels of
ETC complexes were similar in adult subjects with
autism and the control subjects (Group B). A signifi-
cant increase in the levels of lipid hydroperoxides, an
oxidative stress marker, was also observed in the cer-
ebellum and temporal cortex in the children with
autism. These results suggest that the expression of
ETC complexes is decreased in the cerebellum and the
frontal and temporal regions of the brain in children
with autism, which may lead to abnormal energy
metabolism and oxidative stress. Based on these find-
ings, the significant deficits observed in the levels of
ETC complexes in children with autism may readjust
to normal levels by adulthood.
Second, Palmieri and coauthors (2010) studied mito-
chondrial dysfunction in autism using temporocortical
gray matter from six matched patient-control pairs.
The authors performed postmortem biochemical and
genetic studies of the mitochondrial aspartate/gluta-
mate carrier (AGC), which participates in the aspar-
tate/malate reduced nicotinamide adenine dinucleotide
shuttle and is physiologically activated by calcium
(Ca2+). They found that the AGC transport rates were
significantly higher in tissue homogenates from all six
patients, including those with no history of seizures
and with normal electroencephalograms prior to death.
The increase was consistently blunted by the Ca2+
chelator ethylene glycol tetraacetic acid. In addition,
neocortical Ca2+ levels were significantly higher in all
six patients, and no difference in AGC transport rates
was found in isolated mitochondria from patients and
controls following removal of the Ca2+-containing
postmitochondrial supernatant. Expression of AGC1,
the predominant AGC isoform in brain, and cyto-
chrome c oxidase activity were both increased in the
patients diagnosed with ASD, indicating an activation
of mitochondrial metabolism. Furthermore, oxidized
mitochondrial proteins were markedly increased in
four of the six study patients. The authors stated that
excessive Ca2+ levels are responsible for boosting AGC
activity, mitochondrial metabolism and, to a more
variable degree, oxidative stress in the brains of those
As mentioned previously, mercury disrupts calcium
homeostasis in the CNS, and as just mentioned exces-
sive Ca2+ levels are involved in the mitochondrial dys-
function in autism. The following section provides
more evidence for Ca2+ disruption as a product of mer-
cury exposure and evidence of this disruption in the
brains of those with ASD.
Section summary statement
Mercury intoxication can result in mitochondrial
dysfunctionin the brain involving increased mitochon-
drial levels of calcium, which is what is found in the
brains of those with ASD.
Evidence of disruption in calcium homeostasis
and signaling in the cnS from Hg exposure
Calcium is involved in several activities in the CNS
such as transmitter release, long-term potentiation,
growth cone motility, ion channel inactivation, neu-
ronal growth, differentiation, motility and excitability,
secretion of neurotransmitters and hormones, synaptic
plasticity, neurotoxicity and neuronal gene expression,
and calcium signaling (Cote and Crutcher 1991,
Lohmann 2009, Lozac 2010). Optimum levels of cal-
cium influx promote normal dendritic and axonal
elongation and growth cone movements (Lozac 2010).
One reason that calcium homeostasis is critical in the
CNS is because an imbalance of calcium, such as an
excessive influx of Ca2+ ions into the neuron, can lead
to inflammation, free radicals, and ultimately cause
neuronal cell death (Cote and Crutcher 1991). Calcium
signaling (i.e. signal transduction mechanisms where
calcium mobilization, from outside the cell or from
intracellular storage pools, to the cytoplasm is brought
about by external stimuli) is important because it
directs structural and functional adaptations in neu-
rons that underlie the establishment of synaptic speci-
ficity (Lohmann 2009).
Mercury-induced neurotoxicity includes impair-
ment of intracellular calcium homeostasis (Sirois and
Atchison 2000, Castoldi et al. 2003, Atchison 2005,
Ceccatelli et al. 2010) and calcium signaling (Limke et
al. 2004). Calcium homeostasis is disrupted mainly
because mercury induces Ca2+ increases via Ca2+ influx
from the extracellular space (Olanow and Arendash
1994, Liu et al. 2007). Examples of evidence are as
Mercury and the brain pathology in autism 125
Marty and Atchison (1997) used cell imaging and
the Ca2+-sensitive fluorophore fura-2 to investigate the
methylmercury’s effect on Ca2+ homeostasis in rat cer-
ebellar granule cells, that are preferentially targeted by
methylmercury. In vitro methylmercury exposure
(0.2–5.0 μM) induced a biphasic rise in fura-2 fluores-
cence ratio, consisting of a small first phase due to Ca2+
release from intracellular stores and a much larger
second phase which required Ca2+ influx to the cell.
As mentioned in an earlier section on oxidative stress,
Gassó and coauthors (2001) studied the involvement of
oxidative stress and Ca2+ homeostasis disruption in
Hg-induced cytotoxicity on rat cerebellar granule neu-
ron cultures using MeHgCl and HgCl2, and found that,
after 24 hours of exposure, there was a Hg-mediated
(Ca2+) rise. The results of the study indicated that Ca2+
influx through Ca2+ channels and the Na+/Ca2+exchanger
and Ca2+mobilization from the endoplasmic reticulum
are involved in mercury-mediated cytotoxicity. The
authors stated that disruption of redox equilibrium and
Ca2+homeostasis contribute equally to HgCl2-mediated
toxicity, whereas oxidative stress is the main cause of
In order to examine the effects of Thimerosal (which
rapidly converts to a mixture of ethylmercury chloride,
ethylmercury hydroxide, and sodium thiosalicylates in
isotonic saline-based biological media), Zieminska and
colleagues (2010) studied neuron viability, intracellu-
lar levels of calcium and zinc, as well as mitochondrial
membrane potential in primary cultures of rat cerebel-
lar granule cells. The study found that Thimerosal
evoked: (1) a decrease in the cells viability, (2) a rise in
the intracellular calcium and zinc concentration, and
(3) decrease in mitochondrial membrane potential.
Ueha-Ishibashi and coauthors (2004) examined the
effects of Thimerosal as compared to methylmercury
on cerebellar neurons dissociated from 2-week-old
rats. Thimerosal and methylmercury at concentrations
ranging from 0.3 to 10 μM increased the intracellular
concentration of Ca2+[Ca2+(i)] in a concentration-de-
pendent manner. The authors stated that the cytotoxic
potency of Thimerosal and methylmercury were simi-
lar. All of these aforementioned studies on rat cerebel-
lar neurons clearly show that several forms of mercury
have detrimental effects on calcium homeostatis in
Limke and colleagues (2004) conducted a review of
the mechanisms underlying the specific targeting of
cells during MeHg poisoning, and stated that the dis-
ruption of [Ca2+](i) regulation occurs through specific
pathways which affect Ca2+ regulation by organelles,
particularly mitochondria and the smooth endoplasmic
reticulum (SER). Cholinergic pathways which affect
[Ca2+](i) signaling also appear to be critical targets,
particularly muscarinic acetylcholine (ACh) receptors
which are linked to Ca2+ release through inositol-1,4,5-
triphosphate [IP(3)] receptors. [Ca2+](i) dysregulation
may also underlie observed alterations in cerebellar
neuron development through interaction with specific
target(s) in the developing axon. However, Atchison
(2005) studied MeHg and stated that his research sug-
gested that it is the mercury-induced release of gluta-
mate, which, coupled with a Hg-induced impairment
of glutamate uptake by astrocytes, could also cause the
Sirois and Atchison (2000) tested the ability of
methylmercury (MeHg) to block calcium channel cur-
rent in cultures of neonatal cerebellar granule cells
using whole-cell patch clamp techniques and Ba2+ as
charge carrier. The results showed that acute exposure
to submicromolar concentrations of MeHg can block
Ba2+ currents carried through multiple Ca2+ channel
subtypes in primary cultures of cerebellar granule
As seen, many studies show mercury neurotoxicity
includes altered calcium homeostasis. Evidence also
suggests that calcium homeostasis is altered in autism,
which is the topic of the next section.
Evidence of disruption in calcium homeostasis
and signaling in the cnS in autism
Palmieri and Persico (2010) stated in their review
mentioned earlier that recent evidence from postmor-
tem studies of the brains of those with ASD suggests
abnormalities in mitochondrial function as possible
downstream consequences of dysreactive immunity
and altered Ca2+ signaling. Although the topic is new
and the research just beginning, there is a study men-
tioned earlier in the section on mitochondria that
shows evidence of altered calcium homeostasis in
autism (Palmieri et al. 2010). Palmieri and coauthors
(2010) performed postmortem biochemical and genetic
studies of the temporocortical gray matter from six
matched patient-control pairs to examine the mito-
chondrial aspartate/glutamate carrier (AGC), which
participates in the aspartate/malate reduced nicotin-
amide adenine dinucleotide shuttle and is physiologi-
126 J.K. Kern et al.
cally activated by Ca2+. They found that excessive Ca2+
levels are responsible for boosting AGC activity, mito-
chondrial metabolism and, to a more variable degree,
oxidative stress in autistic brains. The authors stated
that AGC and altered Ca2+ homeostasis play a key
interactive role in the cascade of signaling events lead-
ing to autism.
Section summary statement
Mercury intoxication can result in altered Ca2+
homeostasis, involving increased or excessive calcium
levels, which is also what is found in the brains of
those with ASD.
GABA-ErGIc And GLutAMAtErGIc
Evidence of disruption of GABAergic and
glutamatergic homeostasis from Hg exposure
Glutamate is the most common neurotransmitter in
the brain, and it is excitatory. Gamma-aminobutyric
acid (GABA) is the major inhibitory neurotransmitter
of the brain, second only to glutamate as a major brain
neurotransmitter. In order to maintain normal func-
tioning of the brain, glutamate and GABA homeostasis
is critical, especially glutamate homeostasis, because
an excess of excitatory neurotransmitters can cause
excitotoxicity and neuronal cell death (Mutkus et al.
Many studies provide evidence that Hg-induced
neurotoxicity includes disruptions in the GABA
(GABAergic) and glutamate (glutamatergic) systems
in the CNS (Arakawa et al. 1991, Soares et al. 2003,
Castoldi et al. 2003, Mutkus et al. 2005, Basu et al.
2007, van Vliet et al. 2007). Some examples are as fol-
Beginning with GABA, evidence suggests that one
of the main neurotoxic effects of Hg is due to the inhi-
bition of the rate-limiting GABA synthesizing enzyme,
glutamic acid decarboxylase (GAD). Because GAD is
the rate-limiting enzyme responsible for normal con-
version of glutamate to GABA in the brain, measuring
GAD activity can be used to determine any underlying
abnormalities in the GABAergic system (Fatemi et al.
2002, 2009, 2010, Yin et al. 2007). Basu and col-
leagues (2010), for example, examined GAD activity
and found that Hg disrupts GABAergic systems in
discrete brain regions in captive mink. Importantly,
they found that in vitro studies on cortical brain tissues
revealed that inorganic Hg (HgCl2) and methylmer-
curychloride (MeHgCl) inhibited glutamic acid decar-
boxylase (GAD) activity. The authors stated that the
results show that chronic exposure to environmentally
relevant levels of MeHg disrupts GABAergic signal-
There are several other ways that mercury induces
alterations in GABA homeostasis. Some examples are
Fonfría and coauthors (2001) found that mercury
interacts with the GABA(A) receptor by the way of
alkylation of SH groups of cysteinyl residues found in
GABA(A) receptor subunit sequences in cerebellar
granule cells. Hogberg and colleagues (2010) found a
significant downregulation of the GABA receptor rat
cerebellar granule cells after exposure to methylmer-
Rubakhin and colleagues (1995) examined the
effects of inorganic Hg (HgCl2) on GABA activated
Cl- currents on Lymnaea neurons. They found that Hg
enhanced GABA-evoked Cl- permeability and the
higher the Hg concentrations, the higher the membrane
Narahashi and coauthors (1994) examined the
GABA(A) receptor-chloride channel complex and
found that it is a target site of Hg. Kumamoto and col-
leagues (1986) found that MeHgCl greatly inhibits the
GABA uptake into the satellite cells in the dorsal root
Huang and Narahashi (1997) found HgCl2 potentia-
tion of GABA-induced currents. Importantly, they
showed that HgCl2 potentiation of GABA-induced cur-
rents was use dependent, increasing with the frequency
of channel openings. However, the potentiation was
blocked by cysteine and iodoacetamide suggesting
involvement of sulfhydryl groups in this action (Huang
and Narahashi 1996). As mentioned previously, ionic
and alkyl Hg have a strong affinity for sulfhydryl
Similar to GABA research, many studies report Hg
induced alterations of glutamate homeostasis (Farina
et al. 2003a,b, Aschner et al. 2000, Mutkus et al.
2005). Examples are as follows:
In a recent analysis of the effects of Hg on the glu-
tamatergic system, Aschner and coauthors (2000) for
example, found that MeHg-induced dysregulation of
excitatory amino acid homeostasis. They stated that
Mercury and the brain pathology in autism 127
MeHg induces swelling of astrocytes and specifically
inhibits glutamate uptake in astrocytes. This finding
was also noted by several others (Brookes and Kristt
1989, Danbolt 2001, Castoldi et al. 2003, Morken et al.
Yin and colleagues (2011) found that MeHg treat-
ment significantly decreased (P<0.05) astrocytic [(3)
H]-glutamine uptake at all time points and concentra-
tions. The authors stated that the MeHg-induced
changes in astrocytic [(3)H]-glutamine uptake also
resulted in dissipation of the astrocytic mitochondrial
In addition, MeHg increases glutamate extracellular
levels (Juárez et al. 2002, 2005), increases spontaneous
glutamate release (Reynolds and Racz 1987), and
brings about inhibition of glutamate net uptake. These
changes also appear to be related to the cells ability in
maintain cell viability (Moretto et al. 2005, Morken et
al. 2005). Likewise, Vendrell and coauthors (2007)
reported that methylmercury produces loss of cell
viability, reduced intracellular glutamate content, and
increased lipid peroxidation in cultured cerebellar
granule cells of mice.
Soares and colleagues (2003) stated that Hg induces
[3H]-glutamate binding inhibition. Importantly, stud-
ies have found a significant decrease in N-methyl-D-
aspartate (NMDA) receptor levels following Hg expo-
sure (Basu et al. 2007, 2009, Adams et al. 2010). The
NMDA receptor is a specific type of ionotropic gluta-
Other examples of studies that show a significant
decrease in NMDA receptor levels following Hg expo-
sure are as follows:
Wyrembek and colleagues (2010), for example,
examined Thimerosal and HgCl2 effects on GABA and
NMDA-evoked currents in cultured hippocampal neu-
rons using electrophysiological recordings. Following
exposure for 60–90 min to 1 or 10 μM Thimerosal,
there was a significant decrease in NMDA-induced
currents (P<0.05) and GABAergic currents (P<0.05).
Thimerosal was also neurotoxic, damaging a signifi-
cant proportion of neurons after 60–90 min of expo-
sure (recordings were always conducted in the healthi-
est looking neurons). HgCl2, at concentrations 1 μM
and above, was even more toxic, killing a large propor-
tion of cells after just a few minutes of exposure.
Recordings from a few sturdy cells revealed that
micromolar HgCl2 markedly potentiated the
GABAergic currents (P<0.05), but reduced NMDA-
evoked currents (P<0.05). The authors stated HgCl2 act
rapidly, decreasing electrophysiological responses to
NMDA but enhancing responses to GABA, while
Thimerosal works slowly, reducing both NMDA and
GABA responses. The neurotoxic effects of both mer-
curials are interwoven with their modulatory actions
on GABA(A) and NMDA receptors, which most likely
involve binding to these macromolecules.
In addition, Mutkus and coauthors (2005) showed
that MeHg selectively affects glutamate transporter
mRNA expression. Glutamate transporters provide
glutamate for synthesis of GABA and glutathione
As noted in this section, Hg neurotoxicity includes
disruption of the glutamate and GABA systems. These
systems are found to be aberrant in autism, which is
the topic of the next section.
Evidence of alteration of GABAergic and
Glutamatergic homeostasis in autism
Many studies have demonstrated abnormalities
involving the GABAergic and glutamatergic systems
in the autistic brain (Blatt et al. 2001, Dhossche et al.
2002, Fatemi et al. 2008, 2009, Fatemi 2009). Fatemi
and colleagues (2002, 2009, 2010), for example, con-
ducted several studies on the topic of altered GABA
homeostasis in autism. These studies are as follows:
First, they demonstrated that brain levels of glu-
tamic acid decarboxylase 65 and 67kDa proteins
(GAD65/67) were significantly decreased in cerebel-
lum (GAD65) and parietal cortex in autism (Fatemi et
al. 2002). As mentioned, GAD is the rate limiting
enzyme responsible for normal conversion of gluta-
mate to GABA in the brain. This finding was also
reported by Yip and coauthors (2009), who found a
mean 51% reduction in GAD65 mRNA levels in the
larger labeled cells in the autistic group compared with
the levels in the control group (P=0.009) but not in the
smaller cell subpopulation. And earlier in 2007, Yip
and colleagues reported that GAD67 mRNA level was
reduced by 40% in the autistic group, consistent with
previous reports of alterations in the GABAergic sys-
tem in limbic and cerebro-cortical areas in the brains
of those diagnosed with ASD.
Second, Fatemi and coauthors (2009) investigated
the expression of four GABA(A) receptor subunits
and observed significant reductions in GABRA1,
GABRA2, GABRA3, and GABRB3 in parietal cortex
128 J.K. Kern et al.
[Brodmann’s Area 40 (BA40)], while GABRA1 and
GABRB3 were significantly altered in cerebellum,
and GABRA1 was significantly altered in superior
frontal cortex (BA9). Their results demonstrate that
GABA(A) receptors are reduced in three brain regions
that have previously been implicated in the pathogen-
esis of autism, suggesting widespread GABAergic
dysfunction in the brains of subjects with autism. Blatt
and coauthors (2001) also found that GABA(A) recep-
tors are significantly reduced in autism.
Third, Fatemi and colleagues (2010) sought to verify
their western blotting data for GABBR1 via qRT-PCR
and to expand their previous work to measure mRNA
and protein levels of 3 GABA(A) subunits previously
associated with autism (GABRα4; GABRα5; GABRβ1).
Three GABA receptor subunits demonstrated mRNA
and protein level concordance in superior frontal cor-
tex (GABRα4, GABRα5, GABRβ1) and one demon-
strated concordance in cerebellum (GABBR1). These
results provide further evidence of impairment of
GABAergic signaling in autism.
In addition, Harada and coauthors (2011) examined
the neurotransmitters in the GABAergic/glutamatergic
system in 12 patients with autism and 10 normal con-
trols and found that the [GABA]/[Glu] ratio was sig-
nificantly lower (P<0.05) in the patients with autism
than in the normal controls. The authors stated that
this finding thus suggested a possible abnormality in
the regulation between GABA and glutamate.
Similar to GABA, several more studies suggest dis-
ruptions in glutamate homeostasis in ASD (O’Neill et
al. 2003). Bernardi and colleagues (2011), for example,
investigated cellular neurochemistry with proton mag-
netic resonance spectroscopy imaging [(1)H-MRS] in
brain regions associated with networks subserving
alerting, orienting, and executive control of attention
in patients with ASD. The ASD group showed signifi-
cantly lower glutamate concentration in right anterior
cingulate cortex. Page and coauthors (2006) also used
proton magnetic resonance spectroscopy [(1)H-MRS]
to measure the concentration of brain metabolites of
the amygdala-hippocampal complex and a parietal
control region in adults with ASDs and in healthy sub-
jects. Patients with ASD had a significantly higher
concentration of glutamate/glutamine and creatine/
phosphocreatine in the amygdala-hippocampal region
but not in the parietal region.
DeVito and coauthors (2007) examined cerebral
gray and white matter cellular neurochemistry in
autism with proton magnetic resonance spectroscopic
imaging (MRSI) in 26 males with autism (age 9.8 ± 3.2
years) and 29 male comparison subjects (age 11.1 ± 2.4
years). They found that patients with autism exhibited
significantly lower levels of gray matter glutamate than
the control subjects. The deficits were widespread,
according to the authors, affecting most cerebral lobes
and the cerebellum. The authors stated that these
results suggest widespread reductions in gray matter
neuronal integrity and dysfunction of cortical and cer-
ebellar glutamatergic neurons in patients with autism.
Purcell and colleagues (2001) examined brain sam-
ples from a total of 10 individuals with autism and 23
matched controls, mainly from the cerebellum. The
authors reported that the finding showed evidence for
specific abnormalities in the AMPA-type glutamate
receptors and glutamate transporters in the cerebel-
lum. Corroborating these brain studies which suggest
disrupted GABA and glutamate homeostasis, GABA
and glutamate levels are found to be elevated in the
plasma in autism as compared to controls (Dhossche et
al. 2002, Shinohe et al. 2006).
Section summary statement
Mercury intoxication can result in altered of
GABAergic and glutamatergic homeostasis, involving
various areas of the brain, which is also found in the
brains in those with ASD.
InSuLIn-LIKE GroWtH FActor 1 (IGF-1)
SIGnALInG, MEtHIonInE SYntHASE
Evidence of disruption in insulin-like growth
factor 1 (IGF-1) signaling and methionine
synthase inhibition resulting in adverse effects
on methylation from Hg exposure
Insulin-like growth factor 1 (IGF-1) is a protein hor-
mone similar in molecular structure to insulin. IGF-1
has also been referred to as a “sulfation factor”
(Salmon and Daughaday 1957). It is important in
childhood growth and continues to have effects in
adults. Evidence suggests that IGF-1 in the brain has
neuroprotective effects (Tang et al. 2011). For instance,
Park and coauthors (2011) found that IGF-1 down regu-
lates glial activation and induces expression of an
endogenous growth factor.
Mercury and the brain pathology in autism 129
Methionine synthase is an enzyme that interacts
with vitamin B-12 and folate to regenerate the amino
acid methionine from homocysteine (Gallagher and
Meliker 2011). When methionine synthase activity is
inhibited, this in turn, inhibits methylation reactions
(Deth et al. 2008).
Evidence suggests that Hg disrupts IGF-1 signaling
and methionine synthase activity in the brain (Deth et
al. 2008). Suzuki and coauthors (2004), for example,
found that mRNA expression of the estrogen receptor
and insulin-like growth factor 1, which participate in
osteoblastic growth and differentiation, was less than
the control values after treatment with methylmer-
Waly and colleagues (2004) found that Thimerosal
inhibited both IGF-1- and dopamine-stimulated methy-
lation and eliminated methionine synthase activity in
human neuroblastoma cells. The authors stated that
their findings outline a novel growth factor signaling
pathway that regulates methionine synthase activity
and thereby modulates methylation reactions, includ-
ing DNA methylation. Similarly, Deth and colleagues
(2008) found that Thimerosal interferes with PI3-
kinase-dependent methionine synthase, resulting in
impaired methylation, including DNA methylation that
is essential for normal development.
Pilsner and coauthors (2010) found Hg-associated
DNA hypomethylation in polar bear brains. They
examined 47 polar bears. They found that genomic
DNA methylation level was related to postmortem total-
brain-Hg levels (an inverse association was seen between
these two variables for the entire study population).
Evidence of disruption in IGF-1 signaling,
methionine synthase activity, and methylation
Two studies have examined cerebral spinal fluid lev-
els of IGF-1 in autism. First, Vanhala and coauthors
(2001) studied whether IGF-I levels might be associated
with the development of autism. IGF-I levels were mea-
sured in the cerebral spinal fluid (CSF) of 11 children
with autism (4 females, 7 males) using a sensitive radio-
immunoassay method and compared with levels in 11
control participants (6 females, 5 males). Levels of
IGF-I in the CSF were statistically significantly lower in
the children with autism than in the control children.
Second, Riikonen and colleagues (2006) measured
IGF-1 and -2 from cerebrospinal fluid (CSF) by radio
immunoassay in 25 children with autism and in 16
age-matched comparison children without any disabil-
ity. CSF IGF-1 concentration was significantly lower
in patients with autism than in the comparison group.
The CSF concentrations of children with autism less
than 5 years of age were significantly lower than their
age-matched comparisons. The head circumferences
correlated with CSF IGF-1 in children with autism but
no such correlation was found in the comparison
Children with autism also exhibit evidence of
impaired methylation (James et al. 2004, 2006, Deth et
al. 2008). Deth and coauthors (2008) reviewed the
metabolic relationship between oxidative stress and
methylation, with particular emphasis on adaptive
responses that limit activity of cobalamin and folate-
dependent methionine synthase. They stated that chil-
dren with autism exhibit evidence of oxidative stress
and impaired methylation, which may reflect effects of
toxic exposure on sulfur metabolism.
Section summary statement
Mercury intoxication can disrupt IGF-1 signaling,
methionine synthase activity, and methylation in the
brain. The same abnormalities are also found in
dYSFunctIonS, rEducEd BLood FLoW
Evidence for Hg-induced vascular endothelial
dysfunctions, reduced vascularity and blood
flow in the brain
Endothelial cells (ECs) line the interior surface of
blood vessels throughout the entire circulatory system.
The thin layer of endothelial cells is called the endothe-
lium. The functional integrity of endothelium is criti-
cal for the maintenance of blood flow (Wiggers et al.
In vitro exposure to Hg induces cytotoxicity in
endothelial cells (Kishimoto et al. 1995, Wolf and
Baynes 2007, Golpon et al. 2003, Mazeriket al.
2007a,b). Several studies report that Hg causes vascu-
lar endothelial dysfunction resulting in pathological
changes of the blood vessels (Olczak et al. 2010). As
typical of Hg intoxication, there are multiple underly-
130 J.K. Kern et al.
First, research shows that mercury activates differ-
ent types of vascular endothelial cell (EC) phospholi-
pases. Peltz and coauthors (2009), for example, found
that Hg (HgCl2, Thimerosal, and methylmercury) sig-
nificantly activated vascular endothelial cell (EC)
phospholipase D (PLD) in bovine pulmonary artery
ECs (BPAECs). They stated that the study demon-
strated the importance of calcium and calmodulin in
the regulation of Hg-induced phospholipase D activa-
tion, suggesting mechanisms of mercury vasculotoxic-
ity and Hg-induced cardiovascular diseases for both
the inorganic (ionic) and simple alkyl (ethyl and meth-
yl) mercury compounds evaluated.
Likewise, Hagele and colleagues (2007) found that
HgCl2 (inorganic form), MeHgCl (environmental form),
and Thimerosal (EtHgSAL; pharmaceutical form),
induced phospholipase D (PLD) activation. All the
three different forms of Hg significantly induced the
decrease of levels of total cellular thiols. The authors
stated that the study revealed that Hg induced the acti-
vation of PLD in the vascular ECs wherein cellular
thiols and oxidative stress acted as signal mediators for
the enzyme activation, and that the results underscore
the importance of PLD signaling in Hg-induced
Similarly, Mazerik and coauthors (2007b) reported
that the vascular toxicity stems from inorganic Hg
modulating the activity of the vascular endothelial cell
(EC) lipid signaling enzyme phospholipase A(2)
[PLA(2)], which is an important player in the EC bar-
rier functions. Mazerik and colleagues (2007b) stated
that the results suggest that inorganic Hg-induced
PLA(2) activation through the thiol and calcium sig-
naling and the formation of bioactive AA metabolites
further demonstrated the association of PLA(2) with
the cytotoxicity of inorganic Hg in ECs.
Second, oxidative stress appears to be one of the
underlying mechanisms in the destruction of endothe-
lial cells. Evidence suggests that Hg accumulation can
affect endothelial function by inhibiting NO synthesis
and by increasing ROS and lipid peroxidation (Bautista
et al. 2009). The vascular endothelium is highly sensi-
tive to oxidative stress (Wiggers et al. 2008). Furieri
and coauthors (2011) and Wiggers and colleagues
(2008) analyzed the effects of chronic exposure to low
Hg doses on endothelial function, and both concluded
that Hg promotes endothelial dysfunction of coronary
arteries, as shown by decreased NO bioavailability
induced by increased oxidative stress.
Third, Hg also appears to exhibit vascular toxicity
by disrupting calcium stores. Gericke and colleagues
(1993), for example, found that Thimerosal induced
changes of intracellular calcium in human endothelial
cells. They examined the effects of the -SH oxidizing
agent Thimerosal on the intracellular calcium concen-
tration in single endothelial cells from human umbili-
cal cord vein and found that concentrations higher than
10 μM induced a long lasting increase in intracellular
calcium (Thimerosal opens a pathway for Ca2+ entry
from the extracellular side).
Fourth, Asadi and coauthors (2010) reported that
HgCl2 and Thimerosal can stimulate human mast cells
to release vascular endothelial growth factor (VEGF),
which is also vasoactive and pro-inflammatory. This
also contributes to the Hg-induced EC toxicity.
The end result of the Hg-induced EC toxicity is
pathological changes in the blood vessels. Olczak and
colleagues (2010), for example, examined the effects of
early postnatal administration of Thimerosal (4 i.m.
injections, 12 or 240 μg THIM-Hg/kg, on postnatal
days 7, 9, 11 and 15) on brain pathology in Wistar rats.
Numerous neuropathological changes were observed
in young adult rats which were treated postnatally with
Thimerosal. They included: ischaemic degeneration of
neurons and “dark” neurons in the prefrontal and tem-
poral cortex, the hippocampus and the cerebellum,
pathological changes of the blood vessels in the tempo-
ral cortex, diminished synaptophysin reaction in the
hippocampus, atrophy of astroglia in the hippocampus
and cerebellum, and positive caspase-3 reaction in
It is important to note that decreased cerebral blood
flow appears to be a result of the Hg-induced EC toxic-
ity. For example, Hargreaves and coauthors (1988)
gave MeHgCl to rats in a neurotoxic dose regimen (6
daily doses of 8 mg kg−1 p.o.) and also measured
regional cerebral blood flow using iodoantipyrine. The
authors found that blood flow was reduced throughout
The tragedy in Minamata has also provided evidence
for decrease blood flow in the brain of humans as a
result of Hg exposure. At least two studies have found
that the victims of Minamata had decreased brain
blood flow. Itoh and coauthors (2001) found decreased
cerebellar blood flow in patients with Minamata dis-
ease. Even in patients without cerebellar atrophy, blood
flow was significantly decreased. This finding was
corroborated later by Taber and Hurley (2008).
Mercury and the brain pathology in autism 131
Evidence for vascular endothelial dysfunctions,
reduced vascularity and brain blood flow in
Several studies have shown abnormal platelet reac-
tivity and vascular endothelium activation in children
with autism (Yao et al. 2006), as well as decreased
cerebral blood flow in the brain (Galuska et al. 2002).
Yao and colleagues (2006), for example, evaluated the
vascular phenotype in children with autism by mea-
suring urinary levels of isoprostane F(2alpha)-VI, a
marker of lipid peroxidation; 2,3-dinor-thromboxane
B(2), which reflects platelet activation; and 6-keto-
prostaglandin F(1alpha), a marker of endothelium acti-
vation. These were measured by means of gas chroma-
tography-mass spectrometry in subjects with autism
and in healthy control subjects. The results showed
that, compared to the levels in the control subjects,
children with autism had significantly higher urinary
levels of isoprostane F2α-VI, 2,3-dinor-thromboxane B2,
and 6-keto-prostaglandin F1α. Lipid peroxidation levels
directly correlated with both vascular biomarker ratios.
The authors stated that the results indicate enhanced
oxidative stress, platelet and vascular endothelium
Many studies report decreased cerebral blood flow
(rCBF) (or hypoperfusion) in autism (Zilbovicius et al.
1995, 2000, Wilcox et al. 2002, Ito et al. 2005,
Degirmenci et al. 2008). These studies used positron
emission tomography (PET) and single-photon emis-
sion computed tomography (SPECT) as a means of
examining regional cerebral blood in autism as com-
pared to controls. All the studies consistently found
hypoperfusion in autism as compared to the controls.
Examples are as follows:
Using SPECT, Wilcox and coauthors (2002), for
example, examined blood flow in 14 individuals with
ASD and 14 controls ranging in age from 3 to 37 years
and found significant hypoperfusion in the prefrontal
areas of individuals with ASD as compared to controls
in every case. Other areas of the brain reported to have
decreased blood flow in autism using SPECT include:
(1) prefrontal cortex, medial frontal cortex, anterior
cingulate cortex, medial parietal cortex, and/or ante-
rior temporal cortex (Sasaki et al. 2010); (2) frontal
cortex (Zilbovicius et al. 1995); and (3) bilateral insula,
superior temporal gyri and left prefrontal cortices
(Ohnishi et al. 2000). Ohnishi and colleagues (2000)
also reported that analysis of the correlations between
syndrome scores and regional cerebral blood flow
(rCBF) revealed that each syndrome was associated
with a specific pattern of perfusion in the limbic sys-
tem and the medial prefrontal cortex. They stated that
the perfusion abnormalities seem to be related to the
cognitive dysfunction observed in autism, such as
abnormal responses to sensory stimuli, and the obses-
sive desire for sameness.
Examining cerebral perfusion abnormalities in chil-
dren with autism in a segmental quantitative SPECT
study, Gupta and Ratnam (2009) found generalized
hypoperfusion in all of the cases (n=10) as compared to
controls. The authors stated that frontal and prefrontal
regions revealed maximum hypoperfusion and that
subcortical areas also indicated hypoperfusion.
Galuska and coauthors (2002) reported decreased
blood flow with left-sided dominance was found
bifrontally and bitemporally in the autism (perfusion-
metabolism mismatch). Even in those with high func-
tioning autism, hypoperfusion is reported. Ito and
colleagues (2005), for example, examined regional
cerebral blood flow pattern in subjects with high-
functioning autism and found hypoperfusion.
Using PET, areas that have been reported to show
hypoperfusion in ASD as compared to controls include:
(1) both temporal lobes centered in associative auditory
and adjacent multimodal cortex (Zilbovicius et al.
2000); and (2) temporal lobe (Brunelle et al. 2009). No
studies were found that reported normal perfusion in
the brain in autism.
Section summary statement
Mercury intoxication can result in vascular dys-
function and decreased blood flow in various areas of
the brain. These symptoms are also commonly found
in those with ASD.
AMYLoId PrEcurSor ProtEIn
Evidence of Hg-induced increase in the
expression of the amyloid precursor protein
Several studies show that Hg induces an increase in
the expression of the amyloid precursor protein and the
formation of insoluble beta-amyloid (Mutter et al.
2004, Haley 2005, Monnet-Tschudi et al. 2006, Lozac
2010). Using neuroblastoma cells, Olivieri and coau-
thors (2000), for example, examined the potential
132 J.K. Kern et al.
pathophysiological mechanisms of inorganic Hg
(HgCl2) on oxidative stress, cell cytotoxicity, beta-
amyloid production, and tau phosphorylation. The
authors found that exposure of cells to 50 μg/L (180
nM) HgCl2 for 30 min induces a 30% reduction in cel-
lular GSH levels and the release of beta-amyloid pep-
tide. Olivieri and colleagues (2002) examined the role
of estrogen (beta-estradiol) neuroblastoma cells which
had been exposed to Hg and found that Hg induced
oxidative stress and cell cytotoxicity and increased the
secretion of beta-r 1-40 and 1-42. These negative
effects were blocked by the pretreatment of cells with
beta-estradiol. Importantly, Hock and colleagues
(1998) found that levels of amyloid beta-peptide in
cerebral spinal fluid correlated with Hg blood levels in
adults with Alzheimer’s disease.
Evidence of an increase in the amyloid
precursor protein in autism
Although the studies that have examined this
issue are limited, there is evidence to show excess
amyloid precursor protein in autism. Sokol and
coauthors (2006) examined acetylcholinesterase,
plasma neuronal proteins, secreted beta-amyloid
precursor protein (APP), and amyloid-beta 40 and
amyloid-beta 42 peptides in children with and with-
out autism. Children with severe autism and aggres-
sion expressed secreted beta-amyloid precursor
protein at two or more times the levels of children
without autism and up to four times more than chil-
dren with mild autism. In addition, Ray and col-
leagues (2011) examined plasma samples of
amyloid-β (Aβ) precursor protein-alpha form
(sAPPα), sAPPβ (beta form), Aβ peptides, and
brain-derived neurotrophic factor (BDNF) in 18
control, 6 mild-to-moderate, and 15 severely autis-
tic participants. They found that sAPPα levels are
increased and BDNF levels decreased in the plasma
of patients with severe autism as compared to con-
trols. The authors stated that their study provides
evidence that sAPPα levels are generally elevated in
Section summary statement
Mercury intoxication can result in an increase in
the presence of amyloid precursor protein in the brain
and evidence suggests this is also the case in autism.
GrAnuLE And PurKInJE cELL
Evidence of granule and Purkinje cell
degeneration and loss in the cerebellum from
Numerous studies have shown that mercury damages
granule and Purkinje cells in the cerebellum, with effects
that include: (1) heterotopic location (altered location;
Sakamoto et al. 2002, Carvalho et al. 2008); (2) degenera-
tive changes (Cinca et al. 1980, Bertossi et al. 2004,
Olczak et al. 2010); (3) mercury accumulation (Magos et
al. 1985, Warfvinge 2000); and (4) significant cell loss
(Hua et al. 1995, Sorensen et al. 2000, Youn et al. 2002).
Granule and Purkinje cell degeneration and cell loss has
been found to result from: (1) metallic mercury vapor
(Hua et al. 1995, Sorensen et al. 2000); (2) methylmercury
(Warfvinge 2000, Sakamoto et al. 2002, Eto 2006,
Carvalho et al. 2008); (3) mercuric sulfide (HgS; Youn et
al. 2002); (4) ethylmercury (Cinca et al. 1980) and specifi-
cally Thimerosal (Hornig et al. 2004). Also noted, is
Purkinje cell axonal retraction bulbs from methylmercury
exposure (Hunter and Russell 1954). Importantly,
Warfvinge (2000) found evidence that mercury is trapped
in the cerebellum over a long period of time. In a study
which replicated Minamata disease using the common
marmoset, Purkinje and granule cells in the test subjects
were found to be decreased in number in the cerebellum,
depending on the estimated dose of the methylmercury
compound and the duration of the exposure (Eto 2006).
Evidence of granule and Purkinje cell
degeneration and loss in the cerebellum in
One of the most consistent neurological abnormalities
found in persons with autism is marked Purkinje cell loss
in the cerebellum (as determined by histopathological
postmortem examination) and atrophy of the cerebellar
folia (as determined by in vivo neuroimaging) (Ritvo et al.
1986, Courchesne 1991, 1995, Kemper and Bauman 1993,
Bailey et al. 1998, Lee et al. 2002, Palmen et al. 2004,
Whitney et al. 2008, 2009). According to Ritvo and coau-
thors (1986), for example, the Purkinje cells in the vermis
of the cerebellum were approximately 15 standard devia-
tions below the mean, and approximately 8 standard
deviations below the mean bilaterally in the cerebellar
hemispheres in the subjects with autism, as compared to
Mercury and the brain pathology in autism 133
normal controls. Reduction in granule cell number has
also been noted by histopathological post-mortem exami-
nation in autism (Bauman and Kemper 1984, 1985,
Section summary statement
Mercury intoxication can result in loss of granule
and Purkinje cells in the cerebellum, which is a consis-
tent postmortem histopathological abnormaility found
in the brains of those with ASD.
Evidence of mercury induced increased pro-
inflammatory cytokine levels in the brain
(TNF-α, IFN-γ, IL-1beta, IL-6, IL-8)
Several studies show that mercury exposure increases
cytokine levels peripherally (Noda et al. 2003). For exam-
ple, mercury-exposed gold miners have significantly
higher concentrations of pro-inflammatory cytokines
interleukin-1beta (IL-1beta), tumor necrosis factor-alpha
(TNF-alpha or TNF-α), and interferon-gamma (IFN-
gamma or IFN-γ) in serum as compared to the diamond
and emerald miners (Gardner et al. 2010). Mercury based
dental amalgams are found to increase production of
TNF-α and IFN-γ cytokines (Podzimek et al. 2010).
Nyland and coauthors (2012) found that low dose inor-
ganic mercury induced macrophage infiltration and mixed
cytokine response in the heart during acute myocarditis,
including significantly increased interleukin-12, IL-17,
interferon-γ, and TNF-α levels.
In mice, Kim and colleagues (2003) found that oral
exposure to inorganic mercury altered T lymphocyte
phenotypes and cytokine expression. They stated that
mercury altered the expression of inflammatory cytok-
ines (TNF-α, IFN-γ, and IL-12), c-myc, and major
histocompatibility complex II, in various organs.
Importantly, this same effect is found in the mercury
exposed brain. For example, in a recent study by Curtis
and coauthors (2010), chronic mercury exposure (10 week
mercury exposure of 60 ppm HgCl2 in drinking water) in
the prairie vole resulted in a male-specific increase in
TNF-α protein expression in the cerebellum and hip-
pocampus. Importantly, the cerebellum and hippocampus
are two of the main areas found to be affected in ASD
(Kern and Jones 2006). As mentioned in the section on
microglia, Eskes and coauthors (2002) found in brain cell
cultures that isolated microglia were directly activated by
noncytotoxic MeHgCl treatment and there was also an
increase local IL-6 release. They stated that microglial
cells are directly activated by MeHgCl and that the inter-
action between activated microglia and astrocytes can
then increase local IL-6 release.
Evidence of increased pro-inflammatory
cytokine levels in the brain in autism (TNF-α,
IFN-γ, IL-1β, IL-6, IL-8)
Imbalances in the regulation of pro-inflammatory
cytokines have been increasingly correlated in ASD (Lee
et al. 2010) and, in particular, cytokine alteration of
TNF-α is increased in autistic populations (Cohly and
Panja 2005). A number of studies have shown that TNFα,
IFNγ, IL-1β, and IL-12 were increased in the peripheral
blood of ASD patients (Zimmerman et al. 2005, Molloy
et al. 2006, Ashwood and Wakefield 2006).
Similarly, studies show elevated immune response in
the brains and spinal cords of autistic patients. Li and col-
leagues (2009), for example, showed that proinflammato-
ry cytokines (TNF-α, IL-6 and GM-CSF), Th1 cytokine
(IFN-γ) and chemokine (IL-8) were significantly increased
in the brains of ASD patients compared with the controls.
A study by Vargas and coauthors (2005) demonstrated
that tumor growth factor (TGF)-β1, derived from neuro-
glia, was significantly increased in the middle frontal
gyrus (MFG) of autistic patients, while macrophage
chemoattractant protein (MCP)–1, IL-6 and IL-10 were
increased in the anterior cingulate gyrus (ACG). In addi-
tion, using protein array approach, Vargas and colleagues
(2005) also found that MCP-1, IL-6, IL-8 and IFN-γ were
significantly increased in the cerebrospinal fluid (CSF).
TNFα was also shown to be increased in the cerebral spi-
nal fluid of autistic patients by Chez and coauthors (2007).
Chez and colleagues (2007) stated that the elevation of
cerebrospinal fluid levels of TNF-α was significantly
higher (mean = 104.10 pg/mL) than concurrent serum
levels (mean = 2.78 pg/mL) in all of the patients studied.
They stated that the ratio was significantly higher than the
elevations reported for other pathological states for which
cerebrospinal fluid and serum TNF-α levels have been
simultaneously measured and that this finding may pro-
vide insight into central nervous system inflammatory
mechanisms in autism.
According to Wei and colleagues (2012), a number
of studies showed that cytokines are increased in the
blood, brain, and cerebrospinal fluid of autistic sub-
134 J.K. Kern et al.
jects and that elevated IL-6 in the autistic brain has
been a consistent finding. Importantly, Wei and coau-
thors (2011, 2012) completed two studies that showed
that IL-6 may be involved in autism. First, in 2011,
they showed that IL-6 was significantly increased in
the cerebellum of autistic subjects as compared to age-
matched controls and they found that IL-6 over-ex-
pression in granule cells caused impairment in their
adhesion and migration. Then in 2012, Wei and col-
leagues found that that mice with elevated levels of
IL-6 in the brain display many autistic features,
including impaired cognitive abilities, deficits in learn-
ing, abnormal anxiety traits and habituation, as well as
decreased social interactions. In addition, IL-6 eleva-
tion caused alterations in excitatory and inhibitory
synaptic formations and disrupted the balance of excit-
atory/inhibitory synaptic transmissions and also result-
ed in an abnormal shape, length and distributing pat-
tern of dendritic spines.
Section summary statement
Mercury intoxication can result in elevated proin-
flammatory cytokines, specifically, TNF-α, IFN-γ,
IL-1beta, IL-6, and IL-8, which are also elevated in the
brains of those diagnosed with ASD. Increased level of
these cytokines is postulated as one of the mechanisms
leading to histopathological changes found in autism.
ABErrAnt nucLEAr FActor KAPPA-
LIGHt-cHAIn-EnHAncEr oF ActIVAtEd
B CELLS (NF-κB)
Evidence of mercury induced aberrant nuclear
factor kappa-light-chain-enhancer of activated
B cells (NF-κB)
Nuclear factor kappa-light-chain-enhancer of acti-
vated B cells (NF-κB) is a protein found in almost all
cell types mediating regulation of immune response by
induction of expression of the inflammatory cytokines
and chemokines and establishing a feedback mecha-
nism that can produce chronic or excessive inflamma-
tion (Young et al. 2011). NF-κB activation induces
numerous proinflammatory gene products including
cytokines, cyclooxygenase-2 (COX-2), and inducible
nitric oxide synthase (iNOS) (Park and Youn 2011).
Several studies show that mercury exposure
results in aberrant activation of NF-κB (Valko et al.
2005, Assefa et al. 2011, Pal et al. 2011). For exam-
ple, Park and Youn (2011) found that mercury acti-
vates NF-κB, resulting in the induced expression of
COX-2 and iNOS. They stated that the results sug-
gest that mercury can induce inflammatory dis-
eases by lowering host defense. Korashy and El-Kadi
(2008) found that Hg2+ causes the induction of oxi-
dative stress markers, such as ROS and heme oxy-
genase-1 and the depletion of cellular glutathione
content, associated with NF-κB and AP-1 activa-
Dong and coauthors (2001) examined the effects of
methylmercury chloride on DNA binding activities of
NF-κB in developing rat cerebella and cerebra using
electrophoretic mobility shift assays(EMSAs). They
found that NF-κB I and NF-κB II DNA binding activ-
ities of nuclear protein extracts from rat cerebra
exposed to methylmercury chloride in uterus were
lower than in control groups on postnatal day 3 and 7,
while that from rat cerebella was higher than control
groups. They also found that the greater the level of
methylmercury chloride, the higher the NF-κB DNA
binding activities of nuclear protein extracts.
Evidence of aberrant nuclear factor kappa-light-
chain-enhancer of activated B cells (NF-κB) in
the brains of autistic patients
The neuroinflammation in autism appears to be
strong and chronic. Recent research suggests that
this exaggerated response may be due to aberrant
NF-κB expression in autism, which can produce
chronic and/or excessive inflammation (Young et
al. 2011). A recent study by Young and colleagues
(2011) examined NF-κB in human postmortem
samples of orbitofrontal cortex tissue of autistic
patients as compared to controls. According to the
authors, neurons, astrocytes, and microglia all dem-
onstrated increased extranuclear and nuclear trans-
located NF-κB p65 expression in brain tissue from
ASD donors relative to samples from matched con-
trols. These between-groups differences were
increased in astrocytes and microglia relative to
neurons, but particularly pronounced for highly
mature microglia. Measurement of pH in homoge-
nized samples demonstrated a 0.98-unit difference
in means and a strong (F=98.3; P=0.00018) linear
relationship to the expression of nuclear translocat-
ed NF-κB in mature microglia. Young and col-
Mercury and the brain pathology in autism 135
leagues (2011) summarized that NF-κB is aber-
rantly expressed in orbitofrontal cortex in patients
with ASD, as part of a putative molecular cascade
leading to inflammation, especially of resident
immune cells in brain regions associated with the
behavioral and clinical symptoms of ASD. Their
study provides further evidence of neuroinflamma-
tion that may be categorized as excessive in ASD.
Naik and coauthors (2011) examined NF-κB in
peripheral blood samples of 67 children with autism
and 29 control children using electrophoretic mobility
shift assay (EMSA). They stated that there was a sig-
nificant increase in NF-κB DNA binding activity in
peripheral blood samples of children with autism and
when the fold increase of NF-κB in autism cases
(n=67) was compared with that of controls (n=29),
there was a significant difference (3.14 vs. 1.40,
respectively; P<0.02). They concluded that autism
may arise, at least in part, from an NF-κB pathway
Evidence suggests that the equivalent of a vicious
cycle can occur where microglia produce oxidative
products and then increased intracellular ROS, in turn,
activates a redox-sensitive nuclear factor-κB (NF-κB)
to provoke excessive neuroinflammation. According
to Nakanishi and coauthors (2011), this can result in
memory deficits and the prolonged behavioral conse-
Section summary statement
Mercury intoxication can induce NF-κB activation
which may produce chronic or excessive inflammation
in the brain. Such changes are also found in the brains
of those with ASD.
Sources and levels of mercury
Mercury accumulates in both the body and envi-
ronment, and evidence indicates that background
mercury levels are increasing. Environmental stud-
ies and human studies warn of increasing mercury
levels. According to the United States Department
of Interior/US Geological Survey (2002), the US
has a growing mercury level problem. The problem
is not confined to the United States. A 2009 study
of mercury levels in the oceans led by scientists
from Harvard University and the U.S. Geological
Survey, found that the ocean’s mercury levels have
risen about 30% over the last 20 years, predomi-
nately from industrial emissions (Sunderland et al.
2009). The authors stated that mercury is circulated
over vast distances and that Asia’s burning of coal
is the primary source of mercury emissions world-
These increased levels of mercury in the environ-
ment are reflected in human studies. Laks (2009),
for example, recently reported on time trends of
blood inorganic mercury (I-Hg) levels in 6 174
women of ages 18–49, in the National Health and
Nutrition Examination Survey (NHANES) 1999–
2006 data sets. Laks found that in the US popula-
tion, proportion of I-Hg detection rose sharply from
2% in 1999–2000 to 30% in 2005–2006. In addi-
tion, the population averaged mean I-Hg concentra-
tion rose significantly over that same period from
0.33 to 0.39 μ/L. I-Hg was significantly associated
with age suggesting bio-accumulation. Laks (2009)
stated that the study provided evidence that I-Hg
deposition within the human body is a cumulative
process, increasing with age and in the population
over time, since 1999, as a result of chronic mer-
If blood mercury levels in women are increasing,
as suggested by the NHANES data, then it is safe
to assume that fetal exposure to mercury is also
increasing. Studies consistently show that fetal cord
blood mercury levels are higher than the maternal
blood levels (Nyland et al. 2011, Lederman et al.
2008). For example, Nyland and coauthors (2011)
found that fetal cord blood mercury levels were
higher (1.35 times) than in their mother’s blood.
The mercury levels in mother and child were highly
correlated (correlation coefficient of r=0.71; 95%).
They also found that the maternal and fetal cord
blood mercury levels were positively correlated
with inflammatory cytokines (IL-1β, IL-6, and
TNF-α). Lederman and colleagues (2008) exam-
ined levels of total mercury in cord and maternal
blood in US women and found that the total mer-
cury level in the fetal cord blood was over twice the
levels in the blood of the mothers. The study showed
that 1 in 3 US infants has a cord blood total mer-
cury level above the Environmental Protection
Agency (EPA) limit that is considered safe, whereas
one in six mothers of these same children had blood
136 J.K. Kern et al.
mercury levels above the EPA limit. After control-
ling for fish/seafood consumption and other con-
founders, long-term follow-up revealed that the
logarithm of the cord blood mercury was inversely
associated with the Bayley Scales of Infant
Development psychomotor score at 36 months, and
with the Performance, Verbal, and Full IQ scores on
the Wechsler Preschool and Primary Scale of
Intelligence, Revised (WPPSI-R), at 48 months.
Importantly, the maternal blood levels for total
blood mercury in the Lederman and other (2008)
study (where developmental consequences in the
children were noted) were typical of the levels in
the NHANES data representative of women across
the country. Furthermore, the maternal blood mer-
cury level was within the EPAs safety limit for
mercury. Several studies besides the Lederman and
coauthors (2008) study have also shown that even
though the mother’s blood level was within the level
considered to be safe by the EPA, there were still
associated decrements in cognitive function in the
children (Jedrychowski et al. 2006, Lederman et al.
2008, Björnberg et al. 2003).
Significantly, the US has seen a dramatic increase in
the rates of neurodevelopmental disorders in children
in general. Neurodevelopmental disabilities in the US
have so dramatically increased in the last decade, that
at a current rate of 1 in 6 children, neurodevelopmental
disorders are now considered common (Boyle et al.
The relationship between mercury body-burden
and exposure in children appears to be measurable.
For example, a study by Kern and colleagues
(2011b) examined urinary porphyrin levels (a bio-
marker of Hg body burden) in American children
(in Texas) as compared to French children (in east-
ern France). These two groups were chosen because
of mercury exposure differences based on two fac-
tors: (1) exposure from Thimerosal in vaccines and
(2) mercury from coal burning plants. In comparing
the relative presence of Thimerosal in vaccines
administered in the United States (US) and France,
Hessel (2003) reported that in 1999 in the US,
Thimerosal was present in approximately 30 differ-
ent childhood vaccines, whereas there were only 2
in France. In the US, Thimerosal is still in the
influenza vaccines recommended for pregnant
women, infants, and children to be given every year
and it is also in the H1N1 vaccine (both of which
are reportedly seldom administered in France). In
regard to coal burning, energy produced from coal
in France in 2003 was 6% versus 51% in the US and
36.5% in Texas (Encyclopedia of the Nations 2007).
Plus, the children in the Texas group lived in an
area that has one plant alone producing about 1 500
lbs of mercury per year which, because of the pre-
vailing winds, is dispersed over the area in which
they reside. The study found that the US children
have a significantly increased body-burden of mer-
cury in comparison to the body-burden of mercury
in the matched French children.
Some sources of Hg exposure include industrial
emissions, Thimerosal in vaccines, high fructose
corn syrup, compact fluorescent light bulbs, fish,
dental amalgam fillings and dental waste, and
emissions from crematories. Several of these sourc-
es have been found to be associated with autism.
Scientists have found a relationship between autism
and (1) Hg emissions from coal burning plants
(Palmer et al. 2006, 2009, Windham et al. 2006), (2)
Thimerosal in vaccines (Geier et al. 2008, Young et
al. 2008, Gallagher and Goodman 2010), and (3)
dental amalgam fillings (Geier et al. 2009d). In
addition, anecdotal reports have suggested a higher
rate of autism near plants that manufacture compact
fluorescent light bulbs.
How the parallels described in this review add
to the evidence that has already been compiled
in the continuing examination of mercury and
This current review of the parallels between Hg
intoxication and the brain pathology in ASD reveals
many parallels. Even though associations are not
proof of causality, these parallels add to the evidence
that is already compiled in ASD. Furthermore, these
parallels argue against theories questioning the role
of mercury in the pathology of autism on the basis
that these pathologies are dissimilar. On the contrary,
these parallels show that the pathology of the brain in
ASD and mercury intoxication is quite similar.
other metals, toxins, and brain insults and ASd
It, it is important to note, however, that other
heavy metals, toxins and insults can cause patho-
Mercury and the brain pathology in autism 137
logical changes similar to those caused by Hg
which should also be taken into consideration.
Particularly lead (Pb) can cause pathological chang-
es similar to pathology caused by Hg (Korashy and
El-Kadi 2008). However, evidence suggests that
mercury is in a class of its own in that it can cause
all of the types of pathology seen in autism. For
example, the authors could find no evidence that
exposure to Pb decreases cerebral blood flow which
is a common finding in autism.
Most of the studies that have examined the rela-
tionship between autism severity and Hg have
found that there is a positive correlation between
Hg body burden and autism symptom severity. This
includes studies that have examined: (1) urinary
mercury levels (Adams et al. 2009), (2) urinary pre-
coproporphyrins levels, a biomarker of Hg toxicity
(Nataf et al. 2006, Geier and Geier 2007, Geier et
al. 2009b, Kern et al. 2010), (3) hair mercury levels
(Holmes et al. 2003, Elsheshtawy et al. 2011,
Lakshmi Priya and Geetha 2011), and (4) nail mer-
cury levels in ASD (Lakshmi Priya and Geetha
2011). Furthermore, direct measures of total mer-
cury levels in cerebellar samples from the brains of
deceased subjects with autism positively correlated
with the level of oxidative stress markers found in
the brain tissue (Sajdel-Sulkowska et al. 2008).
Although most of the research shows a relation-
ship between ASD and Hg, some do find a relation-
ship with both Pb and Hg. For example, Lakshmi
Priya and Geetha (2011) found a significant eleva-
tion (P<0.001) in the levels of toxic metals Pb and
Hg in both hair and nail samples of a group of autis-
tic children when compared to the control group.
The elevation was more pronounced in the more
severely affected children when compared to chil-
dren with moderate to mild symptom severity.
Elsheshtawy and collegues (2011) had similar find-
ings. They found highly significant differences
between the level of Pb, Hg, and copper in the hair
of children with autism compared with controls,
and there was a positive correlation with autism
severity (using the Childhood Autism Rating Scale)
with both Hg and copper, but not Pb (however, the
intelligence quotient had significant negative cor-
relation with the level of Pb in the hair). In addition,
Adams and coauthors (2009) found a positive rela-
tionship between urinary Pb and Hg levels (after
chelation) and ASD severity.
To the authors’ knowledge, no studies have found
elevated Pb levels in ASD without finding concomitant
elevated Hg levels. Taken together these findings sug-
gest synergistic effects, which is the topic of the fol-
Mercury and synergism: its relevance in autism
Synergism (the interaction of discrete elements
or agents such that the total effect is greater than
the sum of the individual effects) is frequently
reported in mercury research. Mercury has been
found to work synergistically with other metals,
toxins, and pathogens (Mutter 2011). For example,
mercury has been found to have synergistic effects
with the following substances: (1) polychlorinated
biphenyls (PCBs; Bemis and Seegal 1999, Roegge
et al. 2004, 2006); (2) dithiothreitol (Hultberg et al.
2001); acetaminophen (Zwiener et al. 1994); (3)
flame retardant PBDE 99 (2,2’,4,4’,5-pentabromo-
diphenyl ether; Fischer et al. 2008); (4) cadmium
(Mohan et al. 1986, Yu et al. 2008); (5) lead and
manganese (Fernández and Beiras 2001, Papp et al.
2006); (6) endotoxin (a product released by the cell
walls of gram negative bacteria; Rumbeiha et al.
2000); (7) ethanol (Turner et al. 1981); and (8) tes-
tosterone (Geier et al. 2010).
Importantly, mercury can also increase suscepti-
bility of affected individuals to bacterial and viral
infections (Shen et al. 2001). Ilbäck and coauthors
(1996), for example, found that viral infections in
mice were made worse in the presence of mercury
(MeHg). Christensen and colleagues (1996) exam-
ined herpes simplex virus type 2 (HSV-2) in mice
and found that HgCl2 “aggravated” the infection.
Ellermann-Eriksen and coauthors (1994) examined
the influence of HgCl2 on resistance to generalized
infection with herpes simplex virus type 2 (HSV-2)
in mice. They found that mercury, by interfering with
the early macrophage-production of cytokines, dis-
abled the early control of virus replication, leading to
an enhanced infection. Bennett and coauthors (2001)
found that concentrations of Hg were significantly
higher in porpoises that died of infectious disease
compared to healthy porpoises that died from physi-
Furthermore, not only does mercury increase
susceptibility of affected individuals to bacterial
and viral infections, several studies show that viral
138 J.K. Kern et al.
infections cause an increase in brain mercury levels
(Ilbäck et al. 2005, 2007, Frisk et al. 2008). Ilbäck
and coauthors, in 2005, found that coxsackie virus
B3 infection in mice triggered a twofold increase in
the concentration of Hg in the brain. Later, Ilbäck
and colleagues (2007), again, found that viral infec-
tion brought about a significant increase in Hg in
the brain. Moreover, the increase in the brain was
positively correlated to a concomitant decrease
(P<0.05) of Hg in serum. Likewise, Frisk and coau-
thors (2008) found that a common viral infection in
mice increased the amount of Hg in the brain by
52% compared to controls.
In addition, the presence of Hg in the brain can
potentially activate the brain’s immune or neuroin-
flammatory response to a peripheral or central
immune trigger. [It is important to note that the
brain’s immune system can be activated by brain
infection and inflammation, as well as, systemic
infection and inflammation (Teeling and Perry
2009).] Potentiation of the brain’s immune response
can result because Hg: (1) lowers GSH levels (which
can cause microglia mediated neurotoxicity; Lee et
al. 2010) and (2) alters NF-κB functioning (which
can produce chronic or excessive neuroinflamma-
tion; Young et al. 2011). Thus, once the brain’s
immune system is triggered, Hg can then potentiate
the type of neurodegeneration that results from sus-
tained immune activation in the brain by promoting
an exaggerated, sustained response (Banks and
Kastin 1991, Banks et al. 1995). As mentioned ear-
lier, sustained neuroinflammation can result in
neuronal damage and loss of synaptic connections
(Gehrmann et al. 1995). This issue of mercury
working together with the brain’s immune system
to bring about acute symptoms and regression fol-
lowing vaccines has been theorized to explain the
connection between Thimerosal-preserved-vaccines
and the risk of autism.
Research evidence suggests that other metals,
toxins, and pathogens may be involved in the
underlying pathology in autism (Rose et al. 2008).
For example, several studies have shown that toxic
metal levels of arsenic, cadmium, and lead are also
different in children with autism as compared to
typically developing children (Kern et al. 2007). In
addition, it has been suggested from previous stud-
ies that viral infections may play a role in the
pathology (Cohly andPanja 2005, Libbey et al.
2005, Fatemi et al. 2008). Atladóttir and coauthors
(2010), for example, investigated the association
between hospitalization for infection in the perina-
tal/neonatal period or childhood and the diagnosis
of ASD in 1 418 152 children (of which 7 379 chil-
dren were diagnosed as having ASD). Children
admitted to the hospital for any infectious disease
displayed an increased rate of ASD diagnosis.
These examples of synergism suggest that mer-
cury can potentiate or work concomitantly with
other possible contributors in the underlying pathol-
ogy in ASD. Rose and colleagues (2008) who found
a genetic susceptibility to mercury and lead in chil-
dren with autism stated that the individual risk of
developmental neurotoxicity with exposure to envi-
ronmentally relevant levels of lead and mercury is
likely to be determined by genetic susceptibility
factors as well as additive interactions with other
environmental pollutants, cumulative dose, and the
developmental stage of exposure.
In 2000, Bernard and coauthors published a
review of the similarities between Hg poisoning
and the symptoms of autism. In their review,
Bernard and colleagues pointed out 79 similarities
between the symptoms of autism and the symptoms
of mercury intoxication. Since that review, many
more studies have become available to allow for the
current comparison of the specific effects of Hg
exposure in the brain and the similar pathological
findings in those diagnosed with ASD (see Table I
for a summary of the parallels between symptoms
of Hg intoxication in the brain and the brain pathol-
ogy in ASD). The combined similarities outlined by
these two reviews are too numerous to be a result of
chance. Although there may be genetic or develop-
mental components to autism, the evidence in this
current review of the brain findings in autism
clearly indicates the reality of brain injury in ASD;
moreover, the brain injury symptoms which charac-
terize autism closely correspond to those seen in
sub-acute Hg intoxication. The evidence presented
in this paper is consistent with mercury being iden-
tified as either causal or contributory, working syn-
ergistically with other compounds or pathogens in
producing the brain pathology observed in those
diagnosed with ASD.
Mercury and the brain pathology in autism 139
Parallels between mercury intoxication in the brain and the brain pathology in ASD
Hg Effects on the CNS Brain Pathology in Autism
Large, long-range axon degenerationLoss of large, loss range axons
Short-range over connectivity
Brain immune response activation
Brain immune response activation
Elevated GFAP Elevated GFAP
Oxidative stress and lipid peroxidation Oxidative stress and lipid peroxidation
Dose-dependent relationship between mercury levels
and oxidative stress
Dose-dependent relationship between mercury levels and
Decreased reduced glutathione levels and elevated
Decreased reduced glutathione levels and elevated oxidized
Mitochondrial dysfunctionMitochondrial dysfunction
Disrupts calcium homeostasis and signaling Disruption in calcium homeostasis and signaling
Inhibits glutamic acid decarboxylase activityDecreased brain levels of glutamic acid decarboxylase and
Disrupts GABAergic and glutamatergic homeostasisDisrupted GABAergic and glutamatergic homeostasis
Inhibits IGF-1 and methionine synthase activity Altered IGF-1 levels and methionine synthase activity
Impairs methylationImpaired methylation
Vascular endothelial cell dysfunction and pathological
changes of the blood vessels
Vascular endothelial cell dysfunction and pathological
changes of the blood vessels
Decreased cerebral/cerebellar blood flow Decreased cerebral/cerebellar blood flow
Increase in the amyloid precursor protein Increase in the amyloid precursor protein
Granule and Purkinje neuron loss in the cerebellumGranule and Purkinje neuron loss in the cerebellum
Increases in pro-inflammatory cytokine levels (TNF-α,
IFN-γ, IL-1β, IL-6, IL-8)
Increased pro-inflammatory cytokine levels (TNF-α, IFN-γ,
IL-1β, IL-6, IL-8)
Aberrant nuclear factor kappa-light-chain-enhancer of
activated B cells (NF-κB)
Aberrant nuclear factor kappa-light-chain-enhancer of
activated B cells (NF-κB)
140 J.K. Kern et al.
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