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
mailto:[email protected]://dx.doi.org/10.1016/j.neuro.2007.09.010http://dx.doi.org/10.1016/j.neuro.2007.09.010mailto:[email protected]


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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

R. Deth et al. / NeuroToxicology 29 (2008) 190201 191
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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.

R. Deth et al. / NeuroToxicology 29 (2008) 190201192


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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.



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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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