**Molecular Aspects of Thimerosal-induced Autism**

**Richard C. Deth, Ph.D.**

Professor of Pharmacology

Northeastern University

Boston, Massachusetts

**Summary**

The developmental disorder autism has both genetic and environmental
origins, and its forty-fold increase during the past two decades
reflects an increased role for environmental factors. It has been
proposed that increased use of vaccines containing the ethylmercury
derivative thimerasol is the major contributing factor. Published
research from my laboratory has revealed that thimerosal is an
exceptionally potent inhibitor of biochemical pathways that transfer
single carbon atoms between molecules. These "methylation" pathways are
critically involved in several important functions including the
regulation of gene expression and the molecular mechanism of attention.
Recent studies from my lab indicate that thimerosal exerts its toxic
effect on methylation by interfering with formation of the active form
of vitamin B12, also known as cobalamin. Dietary B12 must be converted
to methylB12 (methylcobalamin) in order to assist in the transfer of
single-carbon methyl groups from the folic acid pathway by the enzyme
known as methionine synthase. By reducing methylB12 formation,
thimerosal inhibits this enzyme and thereby interferes with methylation
events. Autistic children have abnormal plasma levels of
methylation-related metabolites and exhibit higher frequencies of
genetic mutations that affect this pathway. These genetic risk factors
make them less able to detoxify thimerosal and also increase their
sensitivity to its mechanism of toxicity. In many cases, autism can be
effectively treated by the administration of methylB12 along with other
agents that augment methylation capacity. Taken together, these facts
indicate that increased exposure to thimerosal has combined with genetic
risk factors in a sensitive subpopulation to cause the recent rise in
autism.

**Outline**

1\. The Puzzle of Autism

2\. Physiological and Biochemical Roles of Methylation

3\. Activity of Methionine Synthase

4\. Effects of Thimerosal and Heavy Metals

5\. Autism-associated Metabolic and Genetic Abnormalities

6\. Methylation-related Treatments for Autism

7\. Conclusions

**[1. The Puzzle of Autism]{.underline}**

Autism is a pervasive developmental disorder characterized by deficits
in language, attention, cognition and learning, frequently accompanied
by abnormal behavior including social isolation, repetitive activity and
emotional lability. Severe deficits may be recognized at birth, but a
failure to achieve standard milestones during initial years of life
remains the primary basis of diagnosis in most cases. While the
underlying cause(s) remains obscure for many developmental disorders,
metabolic abnormalities (e.g. Lesch-Nyhan Syndrome and adenylsuccinate
lyase deficiency) or impaired methylation-dependent gene silencing
and/or imprinting (Rett and Fragile-X Syndromes) (1-4) suggest
biochemical mechanisms that may be involved. Development disorders can
also be caused by exposure to toxins (e.g. ethanol, in fetal alcohol
syndrome; heavy metals, in lead poisoning) (5,6), although the precise
molecular mechanisms underlying their toxicity are not known. The recent
increase in the incidence of autism has led to speculation that
environmental exposures including vaccine additives (i.e. aluminum and
the ethylmercury-containing preservative thimerosal) might contribute to
the triggering of this developmental disorder (7).

Based upon a high concordance in twin studies, genetic factors are
thought to play an important role in causing autism. However, it is
clear that the recent dramatic rise in autism rates is not caused by a
genetic phenomenon. The more likely scenario is that autism is caused by
the interaction of genetic risk factors with environmental risk factors
and the importance of the environmental factors has increased during the
past twenty years. As illustrated in Fig. 1, the "Puzzle of Autism"
therefore is the challenge of understanding exactly which genes provide
the inborn risk, and which environmental factor(s) is serving as the
trigger. The molecular mechanism at the intersection of genetic and
environmental factors should be capable of accounting for the observed
symptoms of autism, and knowledge of this mechanism should help identify
effective treatments for autism. The findings summarized in this report
indicate that impairment in the biochemical pathways that allow for the
transfer of single carbon groups (i.e. methylaion) is a major factor
contributing to the cause(s) of autism.

![](media/image1.wmf){width="3.625in" height="2.53125in"}

**Figure 1:** Autism is caused by a combination of predisposing genetic
factors and environmental factors that synergize with each other to
cause the symptoms that are typical of this developmental disorder.

**[2. Physiological and Biochemical Roles of Methylation]{.underline}**

Methylation is the process by which a single carbon atom is transferred
from a methyl donor to another molecule, commonly resulting in a change
in the functionality of the recipient molecule. This seemingly mundane
biochemical event is vital to life and to the normal capacities of
developed organisms, including man. Perhaps the most important example
of methylation is the epigenetic regulation of gene expression by DNA
methylation. When DNA is methylated, gene expression is suppressed, and
at any one time only a portion of genes are "on" with the others being
turned "off". Since all cells possess the same DNA, differences between
cell types (e.g. neurons *vs.* heart muscle *vs.* liver cells) are due
to specific patterns of DNA methylation that characterize each type.
Development begins with undifferentiated cells (i.e. stem cells) that
gradually assume the characteristics of their final destiny as guided by
sequential shifts in their DNA methylation. Based upon this perspective,
it is easy to see how abnormal methylation could alter the pathway of
normal development and could contribute to neurodevelopmental disorders
such as autism. Indeed, abnormal DNA methylation has previously been
implicated as an important causative factor in Rett and Fragile-X
syndromes (3,4)

As illustrated in Fig. 2, the major methyl donor in biological reactions
is S-adenosylmethionine (SAM), an activated form of the essential,
sulfur-containing, amino acid methionine. After donating its methyl
group, the residual portion of SAM, S-adenosylhomocysteine (SAH), serves
as a regulator of methylation by competing with SAM and inhibiting its
methyl donation. The concentration ratio of \[SAM\]/\[SAH\] therefore
reflects the potential for methylation, and any increase in \[SAH\] or
decrease in \[SAM\] will lower methylation. As described below, children
with autism have low levels of SAM and elevated levels of SAH,
indicating an impaired potential for methylation. Methylation of
neurotransmitters such as dopamine and serotonin terminates their
signaling activity, which may also play a role in autism.

![](media/image2.wmf){width="4.375in" height="1.6472222222222221in"}

**Figure 2:** DNA methylation is carried with S-adenosylmethionine (SAM)
serving as the methyl donor. The resulting S-adenosylhomocysteine (SAH)
inhibits methylation by competing with SAM.

Availability of the methyl donor SAM is critical for methylation. SAM is
formed by addition of an adenosyl group from the high energy molecule
ATP to methionine, as a part of the methionine cycle illustrated in Fig.
3. After methyl donation the adenosyl group is removed from SAH, in a
reversible reaction yielding homocysteine (HCY) and adenosine. Any
unusual build-up of adenosine can shift this reaction backwards toward
SAH formation, while lowering HCY levels. As described below, this
occurs in many children with autism. Activity of the vitamin
B12-dependent enzyme methionine synthase converts HCY back to
methionine, using a methyl group from the folate pathway.

![](media/image3.wmf){width="4.375in" height="3.8652777777777776in"}

**Figure 3:** The four-step methionine cycle involves activation of
methionine (MET) by ATP-dependent adenosylation, methyl donation by SAM,
reversible dissociation of SAH, and remethylation of homocysteine (HCY)
to MET by the vitamin B12-dependent enzyme methionine synthase, using
methylfolate (5-methylTHF) as the methyl donor. HCY can alternatively be
converted to cysteine and glutathione.

The methionine cycle is also involved in the ability of the
neurotransmitter dopamine to stimulate methylation of phospholipids in
the neuronal membrane. This unique process was only discovered several
years ago and its precise function remains unclear at this time.
However, dopamine-stimulated phospholipid methylation (PLM) appears to
be involved in the molecular origins of attention. Genetic variations in
the D4 subtype of dopamine receptor that carries out PLM have been
linked to attention-deficit hyperactivity disorder (ADHD) (8), and the
ADHD-linked variant form is weak in its ability to carry out methylation
(9). Impaired attention is a cardinal symptom of autism, and it is
possible that this reflects reduced activity of dopamine-stimulated PLM.
During dopamine-stimulated PLM, a methionine that is an integral part of
the D4 receptor protein is converted to SAM, then SAH, then HCY and back
to methionine again, as in the methionine cycle of Fig. 3. Thus enzymes
in the methionine cycle, such as methionine synthase, actually have two
substrates, one being a small individual amino acid, and the other being
the large D4 dopamine receptor protein.

[**3.** **Activity of Methionine Synthase**]{.underline}

Methionine synthase is situated at the intersection of the single-carbon
folate pathway and the methionine cycle (Fig. 3), and is therefore
well-positioned to regulate methylation. Its activity serves to maintain
a low level of HCY, limiting its backward conversion to SAH and thereby
promoting methylation. In a recently published study (10), we showed
that methionine synthase activity in cultured human neuronal cells is
substantially stimulated by both dopamine and insulin-like growth
factor-1 (IGF-1) (Table 1). IGF-1 mediates many of the effects of growth
hormone and is a key regulator of development, as well promoting
neuronal myelination.

The mechanism of methionine synthase activation involves an
intracellular signaling pathway, the PI3-kinase pathway, commonly
activated by many different cellular growth factors, including those
that promote cellular differentiation and development. In subsequent
investigations we found that methionine synthase activity in neuronal
cells is absolutely dependent upon the ability of this signaling pathway
to promote the formation of the biologically active form of vitamin B12
(i.e. methylB12 or methylcobalamin). It is pathway that is inhibited by
thimerosal.

![](media/image4.wmf){width="3.8305555555555557in" height="4.875in"}

**Table 1:** Effects of various agents on methionine synthase activity
in neuronal cells. IGF-1 and dopamine stimulate activity, while the
PI3-kinase inhibitor wortmannin, ethanol, mercury (HgCl~2~), lead
(PbNO~3~) and thimerosal inhibit activity.

In the diet we take in vitamin B12 as its hydroxyl derivative,
hydroxycobalamin, which must be subsequently converted to
methylcobalamin before it can function. Dietary vitamin supplements
provide cyanocobalamin, which again must be converted to
methylcobalamin. Conversion to methylcobalamin can occur either directly
in the enzyme methionine synthase itself, or via the pathway outlined in
Fig. 4. As illustrated, methylcobalamin synthesis requires glutathione
(GSH) and SAM, and levels of each of these metabolites are reduced in
autistic children (see below). Although additional studies are needed to
clarify details, growth factors apparently augment synthesis of the
intermediate glutathionylcobalamin, which is subsequently converted to
methylcobalamin. The resultant higher level of methylcobalamin increases
methionine synthase activity, lowering HCY and SAH levels and increasing
methylation. In support of this mechanism, our published study showed
that IGF-1 and dopamine increase the methylation of both DNA and
membrane phospholipids in conjunction with their activation of
methionine synthase.

![](media/image5.wmf){width="3.9993055555555554in"
height="2.7423611111111112in"}

**Figure 4:** Dietary or multivitamin forms of vitamin B12 (cobalamin)
must be converted to the active methylcobalamin form via a two-step
process requiring glutathione (GSH) and SAM.

As illustrated in Fig. 5 (left), methionine synthase normally contains
four domains: 1. A cobalamin-containing catalytic domain. 2. A
methylfolate-binding domain. 3. A HCY-binding domain. 4. A SAM-binding
domain. During the catalytic cycle, folate and HCY domains alternatively
interact with the cobalt ion in cobalamin, which alternates between
Cob(I) and methylated Cob(III) states. Cob(I) is, however, extremely
unstable, and occasionally it oxidizes to the Cob(II) state,
interrupting folate-dependent HCY methylation. Oxidation is especially
likely when levels of methylfolate are low and the Cob(I) state has to
wait too long to receive a methyl group. Under this circumstance, the
SAM-binding domain, when present, carries out a reductive methylation of
Cob(II), with the auxiliary assistance of methionine synthase reductase.
Thus the SAM-binding domain rescues oxidized cobalamin, allowing
methionine synthase activity to resume. Alternatively, oxidized Cob(II)
can be replaced with a new molecule of methylcobalamin to restart the
enzyme. Thus oxidized cobalamin can either be repaired or replaced, but
replacement places a high demand on methylcobalamin synthesis.

![](media/image6.wmf){width="4.866666666666666in"
height="2.8784722222222223in"}

**Figure 5:** Methionine synthase can exist in both four-domain and
three-domain forms. In the three-domain form, the SAM-binding domain
that rescues oxidized Cob(II) is missing. In cells containing only the
three-domain form, oxidized B12 must be replaced with methylB12 to
resume enzyme activity.

In very recent and as yet unpublished studies, we have found evidence
indicating that methionine synthase also exists with only three domains,
with the SAM-binding domain being absent (Fig. 5, right). This form of
the enzyme lacks the ability to rescue oxidized cobalamin, and therefore
is highly dependent upon the availability of methylcobalamin to sustain
activity. As such, this form of the enzyme is subject to regulation by
growth factors and the PI3-kinase signaling pathway, since they control
the level of methylcobalamin synthesis. The particular human neuronal
cell line we utilized contained only the three-domain enzyme. As a
consequence, its methionine synthase activity and its methylation
activity were tightly and completely under the control of the growth
factors signaling pathway.

What would be the advantage to a cell of having a form of methionine
synthase that could not repair its oxidized cobalamin co-factor? While
we do not conclusively know the answer to this question, we hypothesize
that the absence of the SAM-binding domain may improve the ability of
the enzyme to utilize the D4 dopamine receptor as a substrate, since it
is a larger, more bulky substrate than HCY, and the three-domain form is
more prominent in cells expressing the D4 receptor. If correct, this
would imply that the synthesis of methylcobalamin is of particular
importance in those neuronal cells that express the D4 receptor.
Moreover, toxic agents that impair methylcobalamin synthesis would
particularly affect the methylation function of D4 receptors, and would
therefore cause impaired attention.

**[4. Effects of Thimerosal and Heavy Metals]{.underline}**

As described in our published study, a number of neurodevelopmental
toxins share the ability to potently inhibit methionine synthase
activity and methylation. These include ethanol, which causes fetal
alcohol syndrome, heavy metals such as lead, which causes lead
poisoning, as well as mercury and thimerosal. Fig. 6 illustrates the
dose-dependent inhibition of phospholipid methylation (PLM) by lead and
mercury. It is of particular note that concentrations of lead that
reduce cognitive function (IQ) (6) significantly inhibit PLM.
Thimerosal, which releases ethylmercury, was more than 100-fold more
potent than inorganic mercury at inhibiting methylation (Fig. 7). Ten
days after vaccination with a thimerosal-containing vaccine, the
concentration of ethylmercury in blood is reported to be approximately 8
nM (11). In our study, this concentration produced greater than 50%
inhibition of methylation. Assuming that these blood levels are also
present in the brain, one could reasonably expect that vaccine-derived
doses of thimerosal inhibit methylation in the brain.

![](media/image7.wmf){width="4.0in" height="2.55in"}

**Figure 6:** Mercury and lead potently inhibit the ability of IGF-1 to
stimulate phospholipid methylation in human neuroblastoma cells.

![](media/image8.wmf){width="4.5in" height="3.013888888888889in"}

**Figure 7:** Thimerosal potently inhibits IGF-1-induced phospholipid
methylation. Blood levels found in children ten days after vaccination
produced approximately 50% inhibition.

![](media/image9.wmf){width="4.5in" height="4.316666666666666in"}
Thimerosal, ethanol, mercury and lead also inhibited methionine synthase
activity. As shown in Table 1, enzyme activity (i.e. methylation of HCY)
was undetectable after a 30 min pretreatment with a thimerosal
concentration close to the blood level found after vaccination (10 nM).
Thus inhibition of methionine synthase accounts for the inhibitory
effect of thimerosal on methylation. The toxic effect of thimerosal was
also evident simply by observing the shape of cells, which changed from
their usual spindle shape to a condensed, round shape (Fig. 8).

**Figure 8:** Thimerosal induces a dramatic change in the morphology of
human neuroblastoma cells.

We further investigated the mechanism by which thimerosal inhibits
methionine synthase. As shown in Fig. 9 (bottom), when enzyme activity
was measured in the presence of either hydroxycobalamin or
cyanocobalamin, thimerosal caused almost complete inhibition, however in
the presence of methylcobalamin, thimerosal caused no inhibition.
Furthermore, when activity was measured in the presence of
glutathionylcobalamin and SAM, thimerosal inhibition was again absent,
although when SAM was not added, inhibition was observed. This pattern
indicates that thimerosal inhibits the availability of
glutathionylcobalamin, and that this action is responsible for its
inhibition of methionine synthase and methylation.

![](media/image10.wmf){width="3.7493055555555554in"
height="2.9194444444444443in"}

**Figure 9:** The PI3-kinase inhibitor wortmannin and thimerosal
eliminate the ability of hydroxo- and cyanocobalamin to support
methionine synthase activity. The presence of SAM is indicated by (+).

We also examined the ability of different cobalamins to support
methionine synthase activity after inhibition of PI3-kinase. Treatment
with the selective PI3-kinase inhibitor wortmannin caused a pattern of
absolute dependence on methylcobalamin or its synthesis
(gluthionylcobalamin + SAM) that was identical to the effect of
thimerosal (Fig. 9, top). Since thimerosal and wortmannin produce
identical effects, this data strongly suggests that thimerosal acts by
inhibiting the PI3-kinase signaling pathway. **This is the likely
mechanism by which thimerosal causes autism, and may also be the
molecular basis for its toxic effect on bacteria, fungi that makes it an
effective preservative.**

**[5. Autism-associated Metabolic and Genetic
Abnormalities]{.underline}**

Metabolic and genetic studies of autistic subjects provide a more
complete view of how thimerosal, as an environmental insult, causes
autism. Some of the most compelling information has only recently been
obtained, and we are all indebted to the ongoing work of Jill James,
Jeff Bradstreet, Marvin Boris, Alan Goldblatt, Ted Page, Gene Stubbs and
others.

As described in a recent study by Dr. Jill James (12), the concentration
of each of the individual metabolites in the methionine cycle and the
trans-sulfuration pathway leading to glutathione synthesis is
significantly abnormal in autistic children as compared to normal
controls (Table 2). Notably, methionine and SAM levels are low,
consistent with lower activity of methionine synthase. While a low HCY
level might not be expected, the elevated levels of both SAH adenosine
indicate that HCY is being drawn backwards toward SAH via the reversible
activity of the enzyme SAH hydrolase. Thus an elevated level of
adenosine restricts the availability of HCY for both methionine (and
SAM) synthesis and for the formation of cysteine and glutathione.

![](media/image11.wmf){width="5.990277777777778in"
height="2.6131944444444444in"}

**Table 2:** Metabolites in the methionine cycle and transsulfuration
pathway are abnormal in autism (data from Dr. Jill James).

The 20% lower levels of cysteine and 54% lower levels of glutathione in
autistic children will adversely affect their ability to detoxify and
excrete heavy metals and thimerosal. These two compounds directly bind
inorganic and organic mercury and help direct them to the kidneys for
excretion. As a result, these toxic materials will reach a higher free
concentration in the bloodstream of autistic children, will have an
increased potential for transfer to tissue compartments such as the
brain, and will remain in the body for a significantly longer period of
time, as compared to their counterparts who have normal levels of
cysteine and glutathione. These differences begin to define the
subpopulation of children who are more vulnerable to thimerosal and
heavy metal exposure.

Earlier metabolic and genetic studies provide clues to the cause of the
increased adenosine level in autism. Page and co-workers found 8
to10-fold higher activity of the enzyme that makes adenosine
(5\'-nucleotidase) in subgroup of children (13), while Stubbs and
co-workers found that the enzyme that degrades adenosine (adenosine
deaminase) has lower activity in autistic subjects (14). Genetic studies
have also shown that a polymorphism in the adenosine deaminase that
weakens the enzyme is more common among autistic subjects (15).
Impairment of adenosine deaminase, may result from dysfunctional
interactions with its binding partner, enzyme dipeptidyl peptidase IV.
As illustrated in Fig. 10, these metabolic defects can combine with
thimerosal exposure and other genetic risk factors to inhibit
methylation and cause autism.

There is recent evidence that polymorphisms in genes for methionine
synthase and closely-related enzymes are another source of risk for
autism. For example, there are two well-characterized disabling
polymorphisms in the methylenetetrahydrofolate reductase (MTHFR) gene,
the enzyme that makes methylfolate available to methionine synthase, and
these polymorphisms are more common in autism (16). MTHFR polymorphisms
reduce methylfolate levels, which slows the methylation of Cob(I) and
increases the probability that it will oxidize to Cob (II). As a
consequence, MTHFR polymorphisms increase methylcobalamin demand for the
three-domain form of methionine synthase. A disabling polymorphism in
methionine synthase, in a location that can affect the proportion of
three- *vs.* four-domain enzyme forms, is reported to be six-fold more
prevalent in autistic children (17). Finally, a polymorphism in the
enzyme methionine synthase reductase, which assists in the rescue of
cobalamin, may also be more frequent in autism (18). While other
polymorphisms remain to be discovered, these examples serve as examples
of genetic risks that characterize autistic children, making them more
sensitive to the toxic effect of thimerosal and more prone to develop
autism.

![](media/image12.wmf){width="5.125in" height="2.8652777777777776in"}

**Figure 10:** Decreased activity of adenosine deaminase or increased
activity of 5\'-nucleotidase (5\'-NTase) can increase adenosine levels,
resulting in lower levels of HCY, cysteine and glutathione.

**\
**

**6. Methylation-related Treatments for Autism**

If impaired methylation is important in causing autism, metabolic
interventions that augment methylation should be effective treatments.
More specifically, if thimerosal's inhibition of methylcobalamin
synthesis is important in causing autism, then the administration of
methylcobalamin should significantly improve autism. Indeed, this has
proved to be the case. As first reported by Dr. James Neubrander (19),
injections of methylcobalamin, given once every three days, has brought
about significant improvement in approximately 80% of children with
autism. While the degree of improvement varies, a significant number of
children have improved to the point that they are no longer considered
to be "on the autism spectrum". Areas of particular improvement include
language, attention and social skills, which are hallmark symptoms of
autism. Within the next few months, the M.I.N.D. Institute at the
University of California at Davis School of Medicine is slated to carry
out a controlled study of methylcobalamin effectiveness in autism.

Other methylation-promoting treatments are also proving helpful in
autism. In the metabolic study carried out by Dr. Jill James and
colleagues (12), autistic subjects were treated with folinic acid
(leucovorin), a folic acid derivative that augments levels of
5-methylTHF, along with betaine (trimethylglycine), which feeds methyl
groups to the folate pathway. These two agents normalized most of the
abnormal metabolites listed in Table 2, and this was accompanied by
clinical improvement in autism symptoms. Subsequent addition of
methylcobalamin to this regimen brought about further improvement.

While encouraging, these metabolic interventions do not help many
autistic children, and there is a need for additional treatment
approaches. Moreover, improving methylation capacity is only one
component of the multi-dimensional approach to treating autism. Other
elements such as a gluten-free/casein-free diet, chelation of heavy
metals and intensive behavioral therapy are also important. Additional
metabolic interventions, particularly interventions directed at
normalizing adenosine metabolism may prove fruitful. Clearly further
research is needed, building upon the framework of knowledge about how
genetic and environmental factors can synergize to cause autism.

**7. Conclusions**

Autism is a neurological disorder caused by dysfunctional metabolic
control over methylation reactions, and thimerosal appears to be a
precipitating causative factor in many cases. The methionione cycle and
the trans-sulfuration pathway leading to cysteine and glutathione
synthesis are abnormal in autism. Genetic polymorphisms, present in only
a small subpopulation, represent risk factors for autism. As illustrated
in Fig. 11, some of these genetic factors impair detoxification and
clearance of heavy metals, including thimerosal, and also impair the
capacity for methylation. Delayed clearance of thimerosal further
impairs methylation, including both DNA methylation and
dopamine-stimulated phospholipid methylation, adversely affecting growth
factor-directed development and the capacity for attention,
respectively. Autism can be treated, and some of the most effective
treatments, such as methylcobalamin, act by improving methylation. This
encouraging therapeutic development reinforces the conclusion that
thimerosal does indeed cause autism, and it does this by interfering
with methylcobalamin synthesis. This molecular understanding should lead
to new and improved treatments for autism and should provide a
scientifically sound basis for the removal of thimerosal from all
vaccines.

![](media/image13.wmf){width="4.875in" height="4.1402777777777775in"}

**Figure 11:** Genetic and environmental factors combine to cause
autism.

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