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NeuroToxicology 29 (2008) 190201

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 with genetic risk factors. Autistic children exhibit evidence of oxidative stress and impaired methylation, which may reect 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, detoxication, 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 decits secondary to reductions in the capacity for synchronizing neural networks. # 2007 Elsevier Inc. All rights reserved.
Keywords: Arsenic; Attention; Attention-decit 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. 2. 3. 4. 5. 6. Sulfur metabolism and oxidative stress . . . . . . . . . . D4 dopamine receptor-mediated PLM . . . . . . . . . . Heavy metals, xenobiotics, redox and methylation . . Oxidative stress in autism . . . . . . . . . . . . . . . . . . . Redox/methylation-related genetic factors in autism. A redox/methylation hypothesis of autism . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191 193 194 195 196 197 198 198

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

* 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: r.deth@neu.edu (R. Deth). 0161-813X/$ see front matter # 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.neuro.2007.09.010

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

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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-decit 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 exposure may 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 reecting 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 nal common pathway of toxicity, reecting the summed inuence of diverse environmental insults. This role is no great surprise, since sulfur metabolism 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 also required 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 dopaminestimulated 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 decits 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 (Grifth, 1999). The ratio of reduced and oxidized forms of other thiols, such as cysteine and homocysteine (HCY), also contribute to cellular redox status and can equilibrate with GSH/GSSG, but they are present at much lower concentrations and consequently are less inuential. The status of protein thiols and disuldes is closely inuenced 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 NADPHdependent 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 specic 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 neurons against 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 modications to active site cysteine residues (Obin et al., 1998), providing redox

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Fig. 1. Adaptations of sulfur metabolism to oxidative stress. (Panel A) Methylation and redox buffering activities are equally supported by the methionine cycle and transsulfuration during normal redox conditions. (Panel B) During oxidative stress multiple adaptive mechanisms shift the ux 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.

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-bsynthase (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 sulnate, 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 catalyzing 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 GSH synthesis, and methylation capacity is diminished as a result. Testosterone decreases CBS activity, lowers GSH levels and 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 by promoting 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 methylcobalamin (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 oxygen species (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

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Fig. 2. Structure and function of methionine synthase. (Panel A) Methionine synthase contains ve 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, either via the SAM-binding domain and methionine synthase reductase or via replacement of oxidized Cbl(II), allows enzyme reactivation.

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 inuence of methylation, including genes that promote GSH synthesis and/or alleviate oxidative stress, or otherwise participate in the inammatory 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 pathologic consequences, and the particular pathological manifestation (i.e. the particular oxidative stress-induced disease) may reect an individuals genetic background, reected in his/her pattern of single nucleoside polymorphisms (SNPs). Risk-associated SNPs may alter amino acids in the protein product (e.g. enzyme), inuence transcription efciency 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, increased oxidative stress can be viewed as a condition where certain genetic variations prove useful or harmful. 2. D4 dopamine receptor-mediated PLM Dopamine plays a key role in attention. Among ve 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 signicantly reduced (Chauhan and Chauhan, 2006). The molecular basis for dopamine-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 dopaminestimulated PLM (Fig. 1B). When PE is methylated in response to dopamine, membrane uidity in the D4 receptor microenvironment is increased since methylated PE occupies more space and lipid-packing density

methylated, implying that lower activity of methylenetetrahydrofolate 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 with GSH 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 inuence. 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

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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 inuence 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 ring 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 211fold repeat and 35 different versions of the sequence have been identied, 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, and NF-kB pathways (Oak et al., 2001; Zhen et al., 2001). PLMinduced changes in membrane uidity 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 fourrepeat 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, and that it exhibits positive selection (Wang et al., 2004). The sevenrepeat 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 sevenrepeat allele is also associated with a three- to vefold 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 the increased 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 leadinduced neurological impairment. Performance on an attentionrelated task decreased in proportion to documented blood lead levels, and the level of impairment was signicantly 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 to confer 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 signicantly 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 name for thiols is mercaptans, recognizing their afnity 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 difcult, if not impossible, to identify a specic 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 the thiolate 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 modied forms, including mixed disuldes with glutathione, cysteine and homocysteine, oxided forms including sulfenic acid (SOH), sulnic acid (SO2H), and sulfonic acid (SO3H), or S-nitrosocysteine (SNO). These modications 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 sufcient concentrations, environmental exposures will preferentially affect the most sensitive targets. Analysis of neurological decits as a function of plasma lead concentrations failed to identify a threshold level that could be considered as safe, with cognitive decits still observed at concentrations 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 signicantly lowered by mercury and 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 reect 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 broad inuence on cellular activities, including proliferation, differentiation 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 with the 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 organoderivatives exhibit distinctly different distribution and toxicity proles. 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 (Thomas 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 metalinduced oxidative stress and resultant inhibition of methylation may reect 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 GSH synthesis, 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 signicant 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; Kern and Jones, 2006; McGinnis, 2004) there is mounting evidence of oxidative stress and inammation 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 signicantly reduced, indicating impaired methylation capacity. Cystathionine, cysteine and GSH levels are each reduced along with the GSH/GSSG ratio, reecting 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 proles are conrmed 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 inammatory cytokines and evidence of microglial

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activation microglial activation was observed in post-mortem brain sections indicating the presence of neuroinammation (Vargas et al., 2005). Microglia monitor the local environment and provide a macrophage-like function in the brain, releasing pro-inammatory 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 inuence on sulfur metabolism, but also by promoting microglia-based neuroinammation. 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 identied 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, which can include autism, is caused by expansion of CpG methylation sites in the FMR-1 gene (McConkie-Rosell et al., 1993), and folate deciency 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 membrane surface 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 Rettassociated 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 rst 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 nd a signicant association of C677T or A1298C with autism when each 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 afnity 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 homozygosity 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 conjugates GSH 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) detoxies organophosphate pesticides, 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 dopamine 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 ring 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 hypermethylation 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 during attention (Bartos et al., 2007). Development of parvalbuminexpressing 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 reect 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 dening the risk of autism, although their relative contribution can vary greatly. Genetic factors are sufcient for mutations of ASL, Rett and Angelman/Prader-Willi syndromes, while the occurrence 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 reect a major role for environmental factors, including, but not limited to, heavy metal and xenobiotic exposure. In these cases, genetic factors still dene the at-risk population, but instead of frank mutations, risk arises from combinations of polymorphisms (SNPs) carried by signicant 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

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 fundamental to normal development. Impairment of dopamine-stimulated phospholipid methylation limits frequency-dependent synchronization of neuronal networks, reected as decits 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.

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 DNA methylation, 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 inuence 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 SNPs affecting dopaminergic function (e.g. COMT) and/or the neuronal substrates participating in synchronization (e.g. RELN, MET or NGLN3/4). The risk of autism can theoretically be inuenced 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.

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We hope that our redox/methylation hypothesis promotes improved understanding of the molecular origins of autism. The validity of any hypothesis requires that it account for relevant and previously disparate observations. Our redox/methylation hypothesis does integrate ndings across genetic, biochemical, and neurological domains, but does not explicitly account for all autism observations (e.g. abnormalities in brain size, myelination patterns or serotonin levels). However, it may serve as a useful starting point that can be critically tested and accordingly revised 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 metabolites (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 benet from these treatments. Acknowledgements The authors wish to acknowledge research support to RD provided by SafeMinds, Autism Research Institute, and Cure Autism Now. References
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