deth autism methyl hypoth 09
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Review
How environmental and genetic factors combine to cause autism:A redox/methylation hypothesis
Richard Deth *, Christina Muratore, Jorge Benzecry,Verna-Ann Power-Charnitsky, Mostafa Waly
Department of Pharmaceutical Sciences, Northeastern University, Boston, MA 02468, United States
Received 31 January 2007; accepted 27 September 2007
Available online 13 October 2007
Abstract
Recently higher rates of autism diagnosis suggest involvement of environmental factors in causing this developmental disorder, in concert withgenetic risk factors. Autistic children exhibit evidence of oxidative stress and impaired methylation, which may reflect effects of toxic exposure on
sulfur metabolism. We review the metabolic relationship between oxidative stress and methylation, with particular emphasis on adaptive responses
that limit activity of cobalamin and folate-dependent methionine synthase. Methionine synthase activity is required for dopamine-stimulated
phospholipid methylation, a unique membrane-delimited signaling process mediated by the D4 dopamine receptor that promotes neuronal
synchronization and attention, and synchrony is impaired in autism. Genetic polymorphisms adversely affecting sulfur metabolism, methylation,
detoxification, dopamine signaling and the formation of neuronal networks occur more frequently in autistic subjects. On the basis of these
observations, a redox/methylation hypothesis of autism is described, in which oxidative stress, initiated by environment factors in genetically
vulnerable individuals, leads to impaired methylation and neurological deficits secondary to reductions in the capacity for synchronizing neural
networks.
# 2007 Elsevier Inc. All rights reserved.
Keywords: Arsenic; Attention; Attention-deficit hyperactivity disorder (ADHD); D4 dopamine receptor; Folic acid; Heavy metal; Lead; Mercury; Oxidative stress;
Neuronal synchronization; Pesticide; Phospholipid methylation; Thimerosal; Vitamin B12; Xenobiotic
Contents
1. Sulfur metabolism and oxidative stress. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191
2. D4 dopamine receptor-mediated PLM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193
3. Heavy metals, xenobiotics, redox and methylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194
4. Oxidative stress in autism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195
5. Redox/methylation-related genetic factors in autism. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196
6. A redox/methylation hypothesis of autism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198
During the past several decades the prevalence of autism and
related pervasive developmental disorders in the U.S. has
dramatically escalated to epidemic levels, affecting 3 in 10,000
children in 1970, but 66 in 10,000 in 2002 (Rice et al., 2007).
The possible origins of this increase have been the subject of
considerable public debate (Blaxill, 2004), and advances in
detection and broadening of the diagnostic criteria for autism
have been suggested to play a role (Fombonne et al., 2006),
while genetic factors are clearly important, as indicated by high
concordance rates among twins and siblings (Smalley et al.,
1988). However, genetic factors alone cannot account for an
epidemic that developed in the relatively short period of 1020
Available online at www.sciencedirect.com
NeuroToxicology 29 (2008) 190201
* Corresponding author at: Northeastern University, 312 Mugar, 360
Huntington Avenue, Boston, MA 02115, United States. Tel.: +1 617 373 4064;
fax: +1 617 373 8886.
E-mail address: [email protected] (R. Deth).
0161-813X/$ see front matter # 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.neuro.2007.09.010
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years (Herbert et al., 2006). Thus environmental factors are
very likely to account for the major portion of the increased
prevalence of autism.
Exposure to xenobiotics is an inevitable feature of
contemporary life, driven by an ever increasing number of
threatening chemicals found in air, water and food supplies and
other materials we come in contact with during our daily
routines. Heavy metals, such arsenic, lead and mercury, listed
as the three highest priority hazardous substances by the U.S.
Department of Health and Human Services (http://
www.atsdr.cdc.gov/cercla/05list.html), are of particularly high
concern, since even low levels are associated with neurological
impairments, including attention-deficit hyperactivity disorder
(ADHD) and lower IQ (Lanphear et al., 2005; Trasande et al.,
2005; Wright et al., 2006). Other heavy metals (cadmium,
antimony, manganese, nickel, etc.) exert similar effects. It has
been proposed that rising rates of autism are linked to increases
in vaccine-derived doses of the ethylmercury derivative
thimerosal, although this remains a controversial proposal
(Bernard et al., 2002). Heavy metal and xenobiotic exposuremay also contribute to neurodegenerative disorders including
Parkinsons and Alzheimers diseases (Domingo, 2006;
Mellick, 2006; Zintzaras and Hadjigeorgiou, 2004), indicating
that the human brain is an especially sensitive target.
While individual xenobiotics and heavy metals each produce
a unique constellation of pathological insults reflecting their
individual chemical reactivity, almost all such agents directly or
indirectly impact cellular redox status and associated pathways
of sulfur metabolism (Valko et al., 2005). Indeed, sulfur
metabolism can be considered a final common pathway of
toxicity, reflecting the summed influence of diverse environ-
mental insults. This role is no great surprise, since sulfur meta-bolism has evolved as a primary defense system against stressful
insults, orchestrating a large number of processes to maintain
normal cellular function and survival (Winyard et al., 2005).
Recent studies of sulfur metabolism in children with autism
reveal a pattern of abnormalities indicative of the presence of
oxidative stress and impaired methylation (James et al., 2004,
2006). We previously described the shared ability of a number
of neurodevelopmental toxins, including lead, mercury,
thimerosal and alcohol, to potently inhibit activity of
methionine synthase (MS), the ubiquitous vitamin B12 and
folate-dependent enzyme that converts homocysteine (HCY) to
methionine (Waly et al., 2004). As described below, MS activity
is highly sensitive to oxidative stress. MS activity is alsorequired for dopamine-stimulated phospholipid methylation
(PLM), a unique signaling activity of the D4 subtype dopamine
receptor, that appears to be critical for synchronization of brain
activity during attention (Demiralp et al., 2007; Deth, 2003).
Impaired synchronization is a feature of autism, and a large
body of literature links D4 dopamine receptors to ADHD
(Faraone and Khan, 2006; LaHoste et al., 1996), suggesting that
impaired methylation activity of MS could limit dopamine-
stimulated PLM in autism and ADHD.
Based upon the above, a redox/methylation hypothesis of
autism is advanced, proposing that environmental insults
initiate autism in genetically sensitive individuals by promoting
cellular oxidative stress and initiating adaptive responses that
include reduced methylation activity. Impaired methylation in
turn leads to developmental delay and deficits in attention and
neuronal synchronization that are hallmarks of autism.
1. Sulfur metabolism and oxidative stress
All cellular functions are affected by the prevailing redox
status, and sulfur metabolism plays a central role in maintaining
a redox potential that is favorable for homeostasis. The
cysteine-containing tripeptide glutathione (GSH) serves as the
primary intracellular antioxidant, and is maintained at a
remarkably high concentration (e.g. 525 mM), providing a
reservoir of metabolic reducing equivalents (Akerboom et al.,
1982). The ratio of reduced to oxidized forms of GSH (GSH/
GSSG) can be as high as 100, but when the rate of GSH
oxidation exceeds the rate of its formation, this ratio can be
dramatically reduced, creating a state of oxidative stress
(Griffith, 1999). The ratio of reduced and oxidized forms of
other thiols, such as cysteine and homocysteine (HCY), alsocontribute to cellular redox status and can equilibrate with
GSH/GSSG, but they are present at much lower concentrations
and consequently are less influential.
The status of protein thiols and disulfides is closely
influenced by redox status, and oxidative stress causes
metabolic alterations that can disrupt normal cellular function
and can lead to cell death. Some metabolic consequences of
oxidative stress serve to counteract the condition by increasing
the GSH to GSSG ratio. For example, activity of NADPH-
dependent glutathione reductase can be increased (Hamburg
et al., 1994) and/or GSSG can be exported from the cell in order
to restore redox balance (Suzuki and Sugiyama, 1998).However, de novo GSH synthesis is critical to maintain
adequate levels of GSH and to sustain cellular redox balance.
As outlined in Fig. 1A, intracellular levels of the thiol amino
acid cysteine are rate limiting for GSH synthesis, thus
augmenting cysteine availability is a crucial mechanism by
which cells increase GSH to meet demand. There are three
sources of cysteine: (1) uptake from outside of the cell; (2)
protein catabolism; (3) transsulfuration of HCY. Uptake of
extracellular cysteine is accomplished by specific transport
proteins, and in neurons the primary protein is excitatory amino
acid transporter-3 (EAAT3), so named because it also transports
glutamic acid (glutamate) (Himi et al., 2003; Shashidharan
et al., 1997). Recent studies show that EAAT3 protects neuronsagainst oxidative stress by providing cysteine uptake (Aoyama
et al., 2006), and evidence indicates that EAAT3 activity is
increased by activation of the tyrosine kinase-signaling
pathway (Fournier et al., 2004), implying that neuronal growth
factors can promote neuronal survival by increasing cysteine
uptake and GSH synthesis. Catabolism of proteins is increased
in response to stress, and the released cysteine and methionine
can be utilized for GSH synthesis. The proteasome is the
primary source of intracellular protease activity, cleaving
ubiquitin-tagged proteins to release their amino acids.
Ubiquitin ligase activity is regulated by modifications to active
site cysteine residues (Obin et al., 1998), providing redox
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regulation of proteolysis. However, since cysteine is a rarely
utilized amino acid, increased protein catabolism must be
considered an option of last resort for augmenting GSH
synthesis.
Cysteine is synthesized via transsulfuration (Fig. 1A) and its
availability for GSH synthesis can be increased by diverting
more HCY out of the methionine cycle to transsulfuration.Control of this metabolic branchpoint is a fundamental adaptive
response for regulating cellular redox status. Moreover, relative
activities of methionine synthase (MS) and cystathionine-b-
synthase (CBS) determine the balance between methylation
and redox buffering, and both enzymes are responsive to
cellular oxidative status (Banerjee et al., 2003; Deplancke and
Gaskins, 2002; Ludwig and Matthews, 1997; Persa et al., 2004).
Cysteine dioxygenase (CDO) utilizes molecular oxygen to
convert cysteine to cysteine sulfinate, which is further
metabolized to sulfate and taurine (Fig. 1A), competing with
GSH synthesis for available cysteine. In response to lower
cysteine levels and oxidative stress, CDO degradation by the
ubiquitin/proteasome system is accelerated (Stipanuk et al.,
2004), increasing cysteine availability for GSH synthesis
(Fig. 1B), another adaptive response of sulfur metabolism to
oxidative stress.
CBS is a vitamin B6 (pyridoxal)-dependent enzyme catalyz-
ing ligation of HCY and serine to form cystathionine, which is
subsequently hydrolyzed to cysteine and a-ketobutyrate by
cystathionine-g-lyase (CGL). CBS contains a heme group that
monitors redox status, and its oxidation to the ferric state is
associated with increased activity (Banerjee et al., 2003). CBS
activity is negatively regulated by its carboxy-terminal domain,
but binding of the methyl donor S-adenosylmethionine (SAM)
relieves this inhibition, such that transsulfuration is normally
restricted unless adequate SAM levels are achieved. In response
to oxidative stress, the SAM-binding regulatory domain is
cleaved by a ubiquitin/proteasome-dependent mechanism,
increasing CBS activity and rendering it SAM-independent.
Thus oxidative stress augments transsulfuration to increase de
novo GSHsynthesis, andmethylation capacity is diminished as a
result. Testosterone decreases CBS activity, lowers GSH levelsand increases susceptibility to oxidative stress (Prudova et al.,
2007), which may account for the higher prevalence of autism in
males.
Restricting MS activity promotes HCY diversion toward
GSH synthesis, and acute oxidative stress simultaneously
decreases MS activity and increases CBS activity (Persa et al.,
2004). During evolution different strategies for MS inhibition
have been utilized. In plants (e.g. Arabidopsis) and E. coli, MS
inhibition is accomplished by thiolation, wherein accumulating
GSSG reacts with an active-site cysteine to provide inactivation
(Dixon et al., 2005; Hondorp and Matthews, 2004). In higher
species, including man, oxidative stress rapidly inhibits MS bypromoting oxidation of its cobalamin (vitamin B12) cofactor
(Ludwig and Matthews, 1997). Indeed, the biosynthetic
pathway for cobalamin appears to have evolved as a metabolic
adaptation to an increasingly oxidative environment (Raymond
and Segre, 2006).
The cobalt atom of cobalamins corrin ring, which provides
the essence of MS activity, exists in different oxidation states
during the enzymatic cycle (Fig. 2B). In its Cbl(I) state it
abstracts a methyl group from methylfolate to form methylco-
balamin (MeCbl) with cobalamin in its Cbl(III) state (Evans
et al., 2004). Cbl(I) is regarded as a super-nucleophile and
can readily oxidize to inactive Cbl(II), depending upon whether
or not it encounters an oxidizing molecule (e.g. reactive oxygenspecies (ROS) or electrophilic xenobiotic metabolites) in its
local environment (Liptak and Brunold, 2006). As such, Cbl(I)
serves as an exquisitely sensitive redox sensor for the
intracellular environment, and when it oxidizes, MS activity
is temporarily halted, leading to increased GSH synthesis. The
sensitivity of Cbl(I) to oxidation is restricted by a cap
domain that hovers above the vulnerable cobalt atom while it
awaits the next methyl group from methyl methylfolate
(Bandarian et al., 2002). Cobalamin inactivation is thus
another adaptive mechanism to maintain cellular redox
balance. Notably, the probability of Cbl(I) oxidation increases
when methylfolate levels are low, since it must wait longer to be
Fig. 1. Adaptations of sulfur metabolism to oxidative stress. (Panel A) Methy-
lation and redox buffering activities are equally supported by the methionine
cycle and transsulfuration during normal redox conditions. (Panel B) Duringoxidative stress multiple adaptive mechanisms shift the flux of sulfur resources
toward GSH synthesis, including reduced activity of methionine synthase,
increased activity of cystathionine-b-synthase (CBS) and decreased activity
of cysteine dioxygenase (CDO). Lower methionine synthase activity reduces
methylation, including dopamine-stimulated phospholipid methylation and its
role in attention.
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methylated, implying that lower activity of methylenetetrahy-
drofolate reductase (MTHFR) will promote MS inhibition and
increased GSH synthesis.
Reactivation of MS after Cbl(I) oxidation is accomplished
by converting Cbl(II) to MeCbl. In the classical mechanism
Cbl(II) is reduced to Cbl(I) by methionine synthase reductase
(MTRR), followed by addition of a SAM-derived methyl group
provided by the SAM-binding domain (Bandarian et al., 2002).
In an alternative mechanism that requires Cbl(II) dissociation,
Cbl(II) is converted to hydroxocobalamin, which reacts withGSH to form glutathionylcobalamin that is further converted to
MeCbl in a SAM-dependent reaction (Pezacka et al., 1990).
Since it requires GSH, the latter mechanism is highly attuned to
redox status, assuring that MS will only be reactivated when
GSH levels are adequate.
When MS activity is inhibited by oxidative stress, it not only
reduces methylation of HCY, but also inhibits all methylation
reactions, exerting a broad and powerful influence. HCY
formation from S-adenosylhomocysteine (SAH) hydrolysis
during the methionine cycle is a reversible reaction, and SAH
synthesis from adenosine and HCY is thermodynamically
favored (Ueland, 1982). When MS is inactivated, both HCYand
SAH accumulate, and SAH is a powerful inhibitor of
methylation reactions (Fig. 1B). Thus oxidative stress leads
not only to inhibition of HCY methylation by MS, but also to a
general inhibition of cellular methylation reactions, including
DNA methylation and phospholipid methylation as important
examples. Decreased DNA methylation, such as that induced
by oxidative stress, increases expression of genes under the
negative influence of methylation, including genes that promote
GSH synthesis and/or alleviate oxidative stress, or otherwise
participate in the inflammatory response (Chen and Kunsch,
2004; Fratelli et al., 2005).
While adaptive epigenetic responses may be critical for cell
survival, particularly in the short-term, they also interrupt
normal cellular function, depending upon the intensity and
duration of the oxidative challenge. Transient exposure to
oxidative stressors normally allows sulfur metabolism and
epigenetic patterns to return to normal, reversing adaptive
responses as GSH levels return to homeostatic values. However,
prolonged exposure to heavy metals and xenobiotics can cause
long-lasting adaptive epigenetic responses with pathologicconsequences, and the particular pathological manifestation
(i.e. the particular oxidative stress-induced disease) may reflect
an individuals genetic background, reflected in his/her pattern
of single nucleoside polymorphisms (SNPs). Risk-associated
SNPs may alter amino acids in the protein product (e.g.
enzyme), influence transcription efficiency or otherwise affect
the role of the gene, but are distinct from de novo mutations in
that they occur in 1% or more of the population, and contribute
to normal diversity. Thus increased exposure to environmental
stressors places an entire population at risk, but genetically
vulnerable subpopulations are most likely to manifest a
particular disorder, such as autism. In this regard, increasedoxidative stress can be viewed as a condition where certain
genetic variations prove useful or harmful.
2. D4 dopamine receptor-mediated PLM
Dopamineplaysa key role in attention. Among five dopamine
receptor subtypes, the D4 receptor has the unique ability to
transfer folate-derived methyl groups to the plasma membrane
phospholipid phosphatidylethanolamine (PE), a process known
as dopamine-stimulated phospholipid methylation or PLM
(Sharma et al., 1999; Zhao et al., 2001). Levels of PE in
erythrocytes of autistic children are significantly reduced
(Chauhan and Chauhan, 2006). The molecular basis fordopamine-stimulated PLM lies in a methionine residue
(MET313), unique to the D4 receptor, participating in a
methylation cycle paralleling the methionine cycle (Fig. 1A).
However, while the methionine cycle utilizes methionine as a
source of methyl groups, dopamine-stimulated PLM is
absolutely dependent upon methylfolate and MS activity.
Consequently, reductions in MS activity, such as those brought
about by oxidative insults, will directly reduce dopamine-
stimulated PLM (Fig. 1B).
When PE is methylated in response to dopamine, membrane
fluidity in the D4 receptor microenvironment is increased since
methylated PE occupies more space and lipid-packing density
Fig. 2. Structure and function of methionine synthase. (Panel A) Methionine
synthase contains five domains and a cobalamin cofactor. Composite molecular
model based upon structures from Bandarian et al. (2002) and Evans et al.
(2004). Methylfolate and homocysteine domains alternate in transferring a
methyl group to and from cobalamin, while the cap domain partially protects
cobalamin from oxidation while it awaits methylation. The SAM-binding
domain provides a methyl group to oxidized cobalamin, reactivating the
enzyme. (Panel B) During primary turnover Cbl(I) is vulnerable to oxidation,
depending upon prevailing levels of reactive oxygen species (ROS) and
electrophilic xenobiotic metabolites. Formation of methylcobalamin, eithervia the SAM-binding domain and methionine synthase reductase or via
replacement of oxidized Cbl(II), allows enzyme reactivation.
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is decreased. Dopamine-stimulated PLM is estimated to reach a
turnover rate of 2050 methylations/sec/receptor (Deth, 2003),
allowing dopamine to rapidly alter local membrane properties.
This biophysical effect serves as a membrane-delimited
signaling mechanism initiated by the D4 receptor that can
influence the activity of nearby membrane proteins. We
proposed that this unique mechanism allows D4 receptors to
modulate the resonance frequency of neural networks during
dopamine-induced attention (Deth, 2003; Deth et al., 2004;
Kuznetsova and Deth, 2007). Consistent with this proposal, D4
receptor activation promotes a shift of neuronal network firing
to gamma frequency during attention (Demiralp et al., 2007),
while D4 receptor blocking drugs reduce gamma frequency
synchronization during attention (Ahveninen et al., 2000), and
interfere with synchronization-dependent learning (Laviolette
et al., 2005). While a role for D4 receptors in attention and
neuronal synchronization is well supported in the literature,
involvement of dopamine-stimulated PLM in these events has
not yet been demonstrated, and the sequence of events outlined
above therefore remains speculative.The human D4 receptor displays a distinctive repeat motif
found only in primates, and there is a remarkable degree of
individual variability in the number and composition of repeats.
A 48 bp sequence in the D4 receptor gene is present as a 211-
fold repeat and 35 different versions of the sequence have been
identified, making it one of the most variable human genes
(Wang et al., 2004). The repeat sequence codes for proline-rich
segments in the receptor that serve as attachment sites for SH3
domain-containing signaling and scaffold proteins (Oldenhof
et al., 1998). Thus the D4 receptor serves as a center for
multiple forms of signal generation, involving not only classical
G protein pathways, but also tyrosine kinase, MAP kinase, andNF-kB pathways (Oak et al., 2001; Zhen et al., 2001). PLM-
induced changes in membrane fluidity can modulate the energy
barrier for conformational motions of integral membrane
proteins, including ion channels or transporters, enzymes and
receptors, and this modulation can alter resonance properties of
neurons and neuronal assemblies, shifting attended information
to gamma frequency (Deth et al., 2004).
Analysis of a large, worldwide sample showed that the four-
repeat D4 receptor allele is most common ($65%), followed by
the seven-repeat ($25%) and two-repeat forms ($5%), although
there are large differences between ethnic groups (Chang et al.,
1996). There is evidence that the seven-repeat allele arose by a
relatively recent mutational event about 50,000 years ago, andthat it exhibits positive selection (Wang et al., 2004). The seven-
repeat allele is associated with increased novelty-seeking
behaviors (Benjamin et al., 1996; Ebstein et al., 1996), and
the level of attention-associated gamma synchrony is greater in
subjects with the seven-repeat allele, as compared to two or four
repeats (Demiralp et al., 2007). However, presence of the seven-
repeat alleleis also associated with a three- to fivefold higher risk
of ADHD (LaHoste et al., 1996; Faraone and Khan, 2006), and
contributes to lower IQ in the ADHD cohort, in conjunction with
a SNP in the dopamine transporter (Mill et al., 2006). We found
that dopamine-stimulated PLM is lower for the seven-repeat
form vs. two- or four-repeat, but the potency of dopamine is
greater and dopamine activation of methionine synthase is
greater for the seven-repeat form of the receptor (Deth et al.,
2004). These differences may be relevant to theincreased ADHD
risk associated with the seven-repeat receptor, but the frequency
of the seven-repeat allele is not increased in autism (Grady et al.,
2005).
Similar to autism, the prevalence of ADHD has markedly
increased during the past several decades, and the 4:1
predominance of males in ADHD is similar to autism. Since
ADHD is associated with elevated plasma levels of lead and
mercury (Braun et al., 2006; Cheuk and Wong, 2006), oxidative
stress and lower MS activity might contribute to its
pathogenesis. Froehlich et al. (2007) examined the interaction
between D4 receptor repeat alleles and the severity of lead-
induced neurological impairment. Performance on an attention-
related task decreased in proportion to documented blood lead
levels, and the level of impairment was significantly greater at
any level of lead for boys lacking the seven-repeat allele, but
not for girls, and not in boys carrying the seven-repeat allele.
Thus the seven-repeat allele of the D4 receptor appears toconfer protection against lead-induced cognitive impairments,
at least in boys, representing an example of a gene-environment
interaction. However, the seven-repeat allele was associated
with significantly poorer performance on a working memory
task for both boys and girls. Additional studies are needed to
clarify what appears to be a complex relationship between D4
receptor genotype, heavy metal sensitivity and gender.
3. Heavy metals, xenobiotics, redox and methylation
The ability of heavy metals to bind to thiol groups and to
disrupt pathways of sulfur metabolism is well established.Indeed, the traditional namefor thiols is mercaptans, recognizing
their affinity for mercury. Sulfur metabolism is important for the
excretion of xenobiotics (e.g. sulfation and formation of
mercapturic acid derivatives) and their oxidized metabolites
contribute to oxidative stress. Since many pesticides and
preservatives function by disrupting redox events, it is not
surprising they should exert similar effects in humans.
Cysteine residues play critical roles in most proteins, so it is
difficult, if not impossible, to identify a specific protein as the
critical target for heavy metal toxicity. Cysteine residues are
common participants in catalysis and transfer reactions, since
the sulfur bond allows adducts to form and subsequently be
released. Heavy metals such as mercury bind tightly to thethiolate anion, and in its divalent state the inorganic mercuric
cation can simultaneously bind two thiolates, increasing its
retention to almost irreversible levels.
Cysteine residues are commonly viewed as simple reduced
thiols (SH); however, under physiological conditions they also
exist as a mixture of modified forms, including mixed disulfides
with glutathione, cysteine and homocysteine, oxided forms
including sulfenic acid (SOH), sulfinic acid (SO2H), and
sulfonic acid (SO3H), or S-nitrosocysteine (SNO). These
modifications play a central role in orchestrating cellular
metabolism, especially during oxidative stress, and binding of
heavy metals to thiol groups disrupts this orchestration.
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While almost all proteins can be inhibited by heavy metals at
sufficient concentrations, environmental exposures will pre-
ferentially affect the most sensitive targets. Analysis of
neurological deficits as a function of plasma lead concentra-
tions failed to identify a threshold level that could be considered
as safe, with cognitive deficits still observed at concentra-
tions below 10 mg/dl (0.5 mM) (Lanphear et al., 2005); In
addition, ADHD has been reported to associated with elevated
plasma mercury levels (0.4 mg/dl or 20 nM) (Cheuk and Wong,
2006). Candidate targets for heavy metal-induced neurological
toxicity should therefore be inhibited at these concentrations or
below. MS-dependent PLM activity in human neuronal cells is
exceptionally sensitive to heavy metals, with IC50 values of 0.5
and 0.1 mM for lead and mercury, and 0.05, 0.04, 0.2 and
0.1 nM for arsenic, cadmium, antimony and thimerosal,
respectively (Waly et al., 2004; Waly and Deth, unpublished
data). Thus neuronal MS activity can be considered a candidate
target for causing heavy metal-associated ADHD, and may also
be a candidate for causing autism.
Cellular levels of GSH are significantly lowered by mercuryand other heavy metals, although the precise cause remains
unclear (Agrawal et al., 2006; Sakurai et al., 2005; Shenker
et al., 1993). However, the decrease in GSH is not associated
with a large increase of GSSG, and therefore cannot be
attributed to a simple shift in redox status, but rather to a
reduction in the total GSH/GSSG pool. This could reflect
decreased GSH synthesis, increased extrusion of GSH or
increased GSH metabolism. Consistent with the latter
mechanism, it has been proposed that binding of divalent
mercury to GSH facilitates cleavage of its gamma-glutamyl
residue (Rubino et al., 2006). As reviewed by Schafer and
Buettner (2001), GSH/GSSG redox status exerts a broadinfluence on cellular activities, including proliferation, differ-
entiation and survival.
Conjugation of xenobiotics to GSH, either directly or
glutathione-S-transferase catalyzed, is a common mechanism
for their metabolism and eventual clearance from the body.
Increased exposure therefore stresses sulfur metabolism and
competes with redox buffering for available GSH. Conversely,
clearance of xenobiotics, as well as heavy metals, is delayed
under oxidative stress conditions, prolonging their residence in
the body and increasing their opportunity to exert toxic effects.
Xenobiotics are substrates for cytochrome P-450 enzymes,
yielding oxidized products including hydroxides, quinones or
epoxides. The latter electrophilic products readily react withthe supernucleophile Cob(I) state of cobalamin, leading to
formation of inactive alkylcobalamin adducts (Watson et al.,
2004). However, GSH-dependent conversion of Cbl(I) to
gluthionylcobalamin protects against alkylation, which may be
important for conserving MS activity in the presence of
xenobiotics. Depleted GSH levels would therefore increase MS
sensitivity to xenobiotics.
Some heavy metals, such as mercury, arsenic and antimony,
are methylated in a biological environment, and their organo-
derivatives exhibit distinctly different distribution and toxicity
profiles. Methylmercury readily crosses the blood brain barrier
and is one of the most potent neurotoxicants known (Sanfeliu
et al., 2003). In the brain methylmercury is de-methylated to
inorganic mercury, which has a very slow clearance rate (i.e.
years). A comparative study in primates showed that
ethylmercury derived from the vaccine preservation thimerosal
releases more inorganic mercury in the brain than is released by
methylmercury (Burbacher et al., 2005). Arsenic is mono- or
di-methylated via a SAM-dependent methyltransferase (Tho-
mas et al., 2007), while antimony is methylated using
methionine as the methyl donor (Dopp et al., 2004). Recent
reports of high arsenic levels in chicken [arsenicals are
administered to increase growth rates], raises concern about its
possible adverse effects on methylation-regulated processes
(Lasky et al., 2004).
The high sensitivity of neuronal tissue to heavy metal-
induced oxidative stress and resultant inhibition of methylation
may reflect lower transsulfuration activity in neurons. Initially
it was reported that neurons lacked cystathionase activity
(Finkelstein, 1990), consistent with very high levels of
cystathionine (Tallan et al., 1958). Neurons are therefore
highly dependent upon cystine and cysteine uptake for GSHsynthesis, and are more vulnerable to heavy metal-induced
oxidative stress. However, functional transsulfuration was
recently demonstrated in cultured neurons and in fetal brain,
including a significant decrease in GSH levels upon inhibition
of cystathionase (Vitvitsky et al., 2006). While additional
studies are required, transsulfuration does appear to occur in
neurons, although cysthathionase activity is limited compared
to other tissues.
4. Oxidative stress in autism
As previously reviewed (Chauhan and Chauhan, 2006; Kernand Jones, 2006; McGinnis, 2004) there is mounting evidence
of oxidative stress and inflammation in autism. Plasma levels of
methionine cycle and transsulfuration metabolites are reported
to be abnormal in autistic individuals (Geier and Geier, 2006;
James et al., 2004, 2006). Adenosine and SAH levels are
increased while HCY, methionine and SAM levels are low,
consistent with reduced MS activity and increased CBS
activity, while the SAM/SAH ratio is significantly reduced,
indicating impaired methylation capacity. Cystathionine,
cysteine and GSH levels are each reduced along with the
GSH/GSSG ratio, reflecting increased oxidative stress.
Elevated HCY levels have also been reported in autism (Pasca
et al., 2006). Supplementation with a combination of betaine(trimethylglycine) and folinic acid (5-formylTHF) normalized
methionine cycle metabolites, but transsulfuration metabolites
remained abnormal (James et al., 2004). Upon further addition
of methylcobalamin, levels of all metabolites, as well as SAM/
SAH and GSH/GSSG ratios returned to normal. If these
abnormal metabolic profiles are confirmed by others, they will
represent a critically important clue to the origins of autism.
Oxidative stress in autism is associated with increased
plasma levels of malonyldialdehyde, urinary levels of fatty acid
and lipid peroxidation biomarkers (Chauhan et al., 2004; Ming
et al., 2005; Yao et al., 2006; Zoroglu et al., 2004). Elevated
levels of inflammatory cytokines and evidence of microglial
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activation microglial activation was observed in post-mortem
brain sections indicating the presence of neuroinflammation
(Vargas et al., 2005). Microglia monitor the local environment
and provide a macrophage-like function in the brain, releasing
pro-inflammatory substances upon activation. In addition,
microglia take-up organic mercury and convert it to the more
toxic inorganic mercury (Charleston et al., 1995), and in
primate cortex, chronic methylmercury exposure leads to a
large increase in activated microglia (Charleston et al., 1994).
Heavy metals can therefore cause oxidative stress in neurons
not only by their direct influence on sulfur metabolism, but also
by promoting microglia-based neuroinflammation.
5. Redox/methylation-related genetic factors in autism
As noted above, genetic risk factors play a critical role in
autism, particularly as they combine with environmental
exposures (for a review see Persico and Bourgeron, 2006) and
a number of mutations and SNPs have been identified that have
special relevance for oxidative stress and impaired methylation.A rare purely genetic form of autism is caused by mutations
affecting the enzyme adenylosuccinate lysase (ASL) (Stone
et al., 1992). ASL is required for de novo purine synthesis, a
pathway associated with a number of inborn errors of
metabolism causing developmental disorders. ASL mutations
divert an extraordinary proportion of folate-derived carbon
atoms toward purine synthesis in an effort to offset impaired
enzyme activity, reducing the availability of methylfolate for
MS. Autism is a prominent feature of Rett syndrome,
commonly caused by mutations in the MeCP2 gene, which
encodes a protein that binds to methylated DNA and promotes
gene silencing (Amir et al., 1999). Fragile-X syndrome, whichcan include autism, is caused by expansion of CpG methylation
sites in the FMR-1 gene (McConkie-Rosell et al., 1993), and
folate deficiency increases fragility at the FMR-1 locus
(Hagerman et al., 1983). Dendritic spine density is reduced
in Fragile-X (Irwin et al., 2000), which may weaken the ability
to modulate neural networks.
Several studies have found an association between autism
and chromosomal defects involving 15q1113, a region subject
to methylation-dependent genomic imprinting containing
genes for a type 3 ubiquitin ligase (UBE3A) (Baker et al.,
1994; Bolton et al., 2004; Bundey et al., 1994). This region also
codes for a translocase (ATP10C) responsible for maintaining
high levels of the phospholipid PE at the inner membranesurface where it serves as substrate for D4 receptor-mediated
PLM (Herzing et al., 2001). Mutations in 15q1113 are linked
to Angelman, Prader-Willi and Rett syndromes in addition to
autism (Thatcher et al., 2005), and knockout of the Rett-
associated MeCP2 gene also results in reduced levels of
UBE3A (Samaco et al., 2005), indicating broad involvement of
this locus in developmental disorders.
Decreased plasma adenosine deaminase (ADA) activity was
first reported in autistic subjects, by Stubbs et al. (1982).
Several studies subsequently reported a higher frequency of a
lesser active ADA allele among autistic subjects from an Italian
kindred (Lucarelli et al., 2002; Persico et al., 2000). Lower
ADA activity leads to adenosine accumulation, increased SAH
levels, decreased HCY levels, and reduced transsulfuration, a
pattern found in autism (James et al., 2004, 2006).
Methylfolate, the primary circulating form of folate, is
transported into cells by the reduced folate carrier (RFC), which
can exhibit a SNP (A80G) associated with elevated levels of
HCY (Chango et al., 2000), whose frequency is increased in
autism (James et al., 2006). Methylfolate is synthesized by
methyltetrahydrofolate reductase (MTHFR) and the MTHFR
gene exhibits two common polymorphisms, C677T and
A1298C. Homozygosity for C677T reduces enzyme activity
and elevates HCY levels, particularly when folate levels are low
(Molloy et al., 1997), while A1298C reduces MTHFR activity,
but without elevating HCY (Friedman et al., 1999). Boris et al.
(2004) found a higher frequency of homozygous and
heterozygous C677T genotypes among autistic subjects
(23% and 56%) vs. controls (11% and 41%), and compound
heterozygotes were also more common among autistic subjects
(25%) than controls (15%). James et al. (2006) did not find a
significant association of C677T or A1298C with autism wheneach was evaluated individually, but they contributed to an
increased risk when combined with other SNPs.
Transcobalamin II (TCN2) facilitates cellular uptake of
cobalamin, and a C776G SNP in TCN2, lowers its affinity for
cobalamin (Miller et al., 2002). Homozygosity for C776G is
associated with lower plasma levels of the transcobalamin::-
cobalamin complex and increased HCY levels, and homo-
zygosity for C776G is more common in autistic children (26%)
vs. controls (16%) (James et al., 2006). Thus intracellular
cobalamin levels are likely to be lower in autism, placing
methionine synthase activity at risk.
Glutathione-S-transferase M1 (GSTM1), which conjugatesGSH to toxic electrophiles, is reduced or absent in individuals
carrying the GSTM1*0 (null) allele, increasing their sensitivity
to xenobiotics (Hung et al., 2004). Two studies have reported an
association between the null allele and autism (Buyske et al.,
2006; James et al., 2006), suggesting that GST*M1 contributes
to the risk of oxidative stress and autism.
Paraoxonase 1 (PON1) detoxifies organophosphate pesti-
cides, and its activity is lower in serum of autistic subjects, in
association with elevated levels of HCY and lower levels of
cobalamin (Pasca et al., 2006). SNPs in the PON1 gene that
lower its activity are more common in autistic subjects in the
U.S., but not in Italian subjects, which corresponds with a much
higher use of organophosphates in the U.S. (DAmelio et al.,2005). PON1 also is responsible for hydrolysis of a reactive
cyclic form of homocysteine, homocysteine thiolactone,
which decreases insulin release and insulin responsiveness in
a redox-dependent manner (Najib and Sanchez-Margalet, 2001;
Patterson et al., 2007).
Catechol-O-methyltransferase (COMT) inactivates dopa-
mine and other catecholamine neurotransmitters and exhibits a
polymorphism (G472A) yielding a V158M substitution in the
protein that lowers enzyme activity three- to fourfold (Lachman
et al., 1996). Homozygosity for G472A is higher in autistics
(26%) vs. controls (16%) (James et al., 2006), although the A
allele is usually associated with increased cognitive abilities
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(Malhotra et al., 2002). An autism-associated decrease in
methylation capacity could synergize with lower activity of the
V158M enzyme to produce a large increase in dopamine levels,
and impaired MS activity may not sustain an adequate supply of
methyl groups to the D4 receptor under these circumstances.
Reelin, a product of the RELN gene, is an extracellular
protease participating in the migration of cortical neurons,
particularly parvalbumin-expressing GABAergic interneurons,
during development and also modulates neuronal firing activity
and long-term potentiation (Beffert et al., 2006; Fatemi, 2005).
Reelin expression is subject to epigenetic regulation by
methylation (Chen et al., 2002), and lower brain levels are
found in autism (Fatemi et al., 2001), suggesting hypermethy-
lation of the RELN locus. Consistent with this relationship,
variants of RELN involving repeat sequences in the 50-UTR are
associated with autism (Persico et al., 2001). D4 dopamine
receptors are abundant in the parvalbumin-expressing
GABAergic interneurons that produce reelin (Mrzljak et al.,
1996), and networks containing these interneurons are
important in generating gamma frequency oscillations duringattention (Bartos et al., 2007). Development of parvalbumin-
expressing interneurons requires hepatocyte growth factor/
scatter factor and its tyrosine kinase-linked receptor MET, and a
recent study found a higher frequency of a SNP that lowers
MET transcription in autistic subjects (Campbell et al., 2006).
Synchronized gamma activity is reduced in autism (Wilson
et al., 2006), which may reflect impaired dopamine-stimulated
PLM in the context of SNPs affecting reelin, MET and other
determinants of interneuron networks. Autism-associated
mutations in neuroligin (NLGN3 and NLGN4) (Laumonnier
et al., 2004), which stabilizes synapses, may also affect
synchronization of neuronal networks.
6. A redox/methylation hypothesis of autism
The preceding observations support a redox/methylation
hypothesis of autism. As summarized in Fig. 3, genetic and
environmental factors both play fundamental roles in defining
the risk of autism, although their relative contribution can vary
greatly. Genetic factors are sufficient for mutations of ASL,
Rett and Angelman/Prader-Willi syndromes, while the occur-
rence of autism in Fragile-X syndrome and other intermediate
examples (e.g. tuberous sclerosis) depends upon additional
genetic or environmental factors. However, most autism cases
arising during the past two decades undoubtedly reflect a majorrole for environmental factors, including, but not limited to,
heavy metal and xenobiotic exposure. In these cases, genetic
factors still define the at-risk population, but instead of frank
mutations, risk arises from combinations of polymorphisms
(SNPs) carried by significant proportions of the human
population. In a particular individual the likelihood and
severity of oxidative stress in response to a potentially toxic
environmental exposure depends upon the presence or absence
of SNPs directly or indirectly affecting sulfur metabolism and/
or other metabolic systems that respond to such exposures (e.g.
PON1, GSTM1*0). The level of MS inhibition and impaired
methylation depends upon the extent of oxidative stress, but
also on SNPs affecting cobalamin and folate status, as well as
SNPs affecting enzymes and metabolites of the methionine
cycle (e.g. MTHFR, RFC, TCN2).
A lower SAM/SAH ratio reduces the probability of DNAmethylation, with consequences for epigenetic regulation of
gene expression and its pivotal role in developmental trajectory,
and SNPs impacting any of the multiple steps leading to gene
silencing or imprinting will influence the severity of disruption.
Since oxidative stress is a systemic feature of autism (James
et al., 2004, 2006), consequences of impaired methylation and
epigenetic disruption will also be expressed in non-neuronal
tissues, giving rise to diverse symptoms such immune or GI
dysfunction, which are commonly seen in autism.
Since D4 receptor-mediate dopamine-stimulated PLM is
absolutely dependent upon MS activity, SNPs promoting
oxidative stress and impaired methylation confer risk to its role
in synchronizing neural networks, synergizing with SNPsaffecting dopaminergic function (e.g. COMT) and/or the
neuronal substrates participating in synchronization (e.g.
RELN, METor NGLN3/4). The risk of autism can theoretically
be influenced by SNPs acting at any level in metabolic and
neuroanatomic pathways supporting neuronal synchronization,
which is essential for complex abilities that are a hallmark of
the human brain. These SNPs have presumably been retained
because they can, in certain circumstances, contribute in a
positive manner to attentive and cognitive abilities. However, in
a more challenging environment, such as increased exposure to
heavy metals and xenobiotics, these same features provide a
source of risk.
Fig. 3. A redox/methylation hypothesis of autism. Environmental factors (e.g.
heavy metals and xenobiotics) can precipitate oxidative stress in a vulnerable
subpopulation possessing risk genes (shown in italics), initiating multiple
adaptive responses involving sulfur metabolism. Inhibition of methionine
synthase broadly reduces methylation activity, with DNA methylation and
dopamine-stimulated phospholipid methylation being important examples.
Reduced DNA methylation interferes with epigenetic events that are funda-
mental to normal development. Impairment of dopamine-stimulated phospho-
lipid methylation limits frequency-dependent synchronization of neuronal
networks, reflected as deficits in attention and cognition. While all cell types
are subject to similar effects, which may be manifested as autism-associated
symptoms, neuronal cells exhibit higher sensitivity to oxidative stress.
R. Deth et al. / NeuroToxicology 29 (2008) 190201 197
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We hope that our redox/methylation hypothesis promotes
improved understanding of the molecular origins of autism. The
validityof any hypothesis requires that it account forrelevant and
previously disparate observations. Our redox/methylation
hypothesis does integrate findings across genetic, biochemical,
and neurological domains, but does not explicitly account for all
autism observations (e.g.abnormalities in brainsize, myelination
patterns or serotonin levels). However, it may serve as a useful
starting point that canbe critically tested andaccordinglyrevised
or even discarded.A useful hypothesis for autism should not only
specify causative factors, but also identify strategies for
treatment. The ability of a regimen of folinic acid, betaine and
methylcobalamin to normalize plasma levels of sulfur metabo-
lites (James et al., 2004) indicates that methylation support and
antioxidant strategies are likely to be useful in treating autism.
Further clinical assessment of these and other therapeutic
approaches is needed in order to validate their utility. It is
reasonable to project that other conditions in which oxidative
stress play a role may also benefit from these treatments.
Acknowledgements
The authors wish to acknowledge research support to RD
provided by SafeMinds, Autism Research Institute, and Cure
Autism Now.
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