Faces of Deth

Please read the Neurodiversity Weblog where Kathleen tells us about an unexpected telephone call from Richard Deth, Professor of Pharmacology at Northeastern University: An Exchange of Views

Kathleen was kind enough to provide a link to my blog entry on Glutathione in a reply to Dr. Deth which he acknowledged with this statement:

"The work of Dr. Jill James is remarkably clear and significant, in my
opinion, and the blog that you reference as a rebuttal is certainly a weak
attempt to ignore important information that points toward a reasonable molecular mechanism. It's as if some people would prefer to not know a cause for autism if it happens to involve mercury or the biochemical systems that are most sensitive to mercury. Go figure."

Well, I guess my feelings should be hurt but I'm not sure if it was meant as an insult or not. I made a weak attempt to ignore important information? Umm....OK, I guess I've done the same thing when a member of a religious organization tries to hand me literature at the airport. Only the truly persistent zealots require a strong attempt to ignore important information.

I thought my blog entry on glutathione was a weak attempt to delineate what is and what is not known about glutathione and methylation in children with autism. It's easy to speculate that mercury may deplete glutathione or interfere with methylation pathways in children with autism but it's quite another to show that either are disrupted as a result of thimerosal content in vaccines.

A better example of "an attempt to ignore important information" might be a journal article that primarily discusses hypothetical in vivo mechanisms that may (or may not) follow a set of in vitro experiments. In fact such a thing may just qualify as a strong and deliberate attempt to ignore what is known about the biochemistry of some children with autism in favor of propping up a pet hypothesis.

Sure thimerosal can deplete GSH at high concentrations. Sure thimerosal can interfere with methylation pathways at various concentrations. Is that evidence that it causes autism? Sure, if you attempt to ignore a large body of literature to the contrary.

Let's take a look at Deth's most recent publication and make a weak attempt to ignore the less important information.

Activation of methionine synthase by insulin-like growth factor-1 and dopamine: a target for neurodevelopmental toxins and thimerosal

The full text is only available by subscription but, like all of the 'important' articles on thimerosal and autism, the full text is freely available online, often before journal publication. How about that. In order to include poorly understood or largely theoretical mechanisms in a scientific publication, an author will sometimes include certain key word qualifiers. We'll call them waffle words and I'll highlight them in green so the reader can attempt to ignore unimportant information with no relevance to autism.

From the abstract:

Methylation events play a critical role in the ability of growth factors to promote normal development.

True. hard to ignore the truth in that statement. Do methylation events play a critical role in the ability of growth factors to promote normal development in children with autism? Possibly but DNA methylation, chromatin remodeling, histone acetylation, etc., all belong to the still emerging field of epigentics. Very little is known about the role of epigenetic factors in the development of autism.

Neurodevelopmental toxins, such as ethanol and heavy metals, interrupt
growth factor signaling, raising the possibility that they might exert adverse effects on methylation.

OK, the possibility has been raised. Now if we knew which, if any, growth factor signaling is interrupted in children with autism this might be relevant.

We found that insulin-like growth factor-1 (IGF-1)- and
dopamine-stimulated methionine synthase (MS) activity and folate-dependent
methylation of phospholipids in SH-SY5Y human neuroblastoma cells, via a
PI3-kinase- and MAP-kinase-dependent mechanism.

This is something that actually adds to our knowledge of the way IGF-1 stimulates MS activity and phospholipid methylation. This would be relevant to autism if the activity of methionine synthase was evaluated in patients with autism and shown to be decreased relative to control subjects. To the best of my knowledge that hasn't happened yet.

The stimulation of this pathway increased DNA methylation, while its
inhibition increased methylation-sensitive gene expression.

This may fit with what we know about some cases of autism once we have a better understanding of which methylation sensitive genes are under or over expressed in certain individuals. Since gene expression and DNA methylation are complex, it is impossible to discuss DNA methylation for an individual in broad terms like increased or decreased. Both can exist in parallel.

Ethanol potently interfered with IGF-1 activation of MS and blocked its
effect on DNA methylation, whereas it did not inhibit the effects of
dopamine.

I'll drink to that! Maybe another reason not to drink while pregnant or nursing. One might argue that autism is similar to Fetal Alcohol Syndrome, or not, but potential epigenetic alterations in neural crest cells would probably happen early in fetal development.

Metal ions potently affected IGF-1 and dopamine-stimulated MS activity, as
well as folate-dependent phospholipid methylation: Cu2+ promoted enzyme activity and methylation, while Cu+, Pb2+, Hg2+ and Al3+ were inhibitory.

Again, an interesting in vitro effect. Relevance to autism? Unknown. If dopamine and IGF-1 levels were normal in autism but MS activity and phospholipid methylation were reduced, metal ion toxicity might be something to consider. Unfortunately, IGF-1 levels are decreased in the CSF of some children with autism, dopamine turnover rates seem normal, methionine synthase activity is unknown, as is phosholipid methylation status. Possibly one out of four parameters.

The ethylmercury-containing preservative thimerosal inhibited both IGF-1-
and dopamine-stimulated methylation with an IC50 of 1 nM and eliminated MS
activity. Our findings outline a novel growth factor signaling pathway that
regulates MS activity and thereby modulates methylation reactions, including DNA methylation.

An in vitro effect that hasn't been demonstrated in vivo let alone in autistic humans.

The potent inhibition of this pathway by ethanol, lead, mercury,
aluminum and thimerosal suggests that it may be an important target
of neurodevelopmental toxins.

Indeed it may. Until we can say that this pathway is inhibited in autism, until we can identify or eliminate other potential factors that influence this pathway, until we develop a suitable animal model to prove the hypothesis, these experiments will require much more than a weak attempt to ignore the hurdles that must be cleared before we can point to a reasonable molecular mechanism.

One seemingly important bit of information is barely touched on in the Waly et al communication. If IGF-1 is required to stimulate methionine synthase activity and intracerebroventricularIGF-1 levels are already low in some cases of autism, couldn't that be sufficient to explain reduced MS activity, assuming it is actually reduced?

"Reduced IGF-1 levels have been reported in autism, which may also contribute to impaired
myelination."

The rather lengthy discussion section of the article is a weak attempt to draw connections between the results of the in vitro experiments and various aspects of autism and miscellaneous neurodevelopmental disorders.

Let's look for those waffle words:

Discussion:
MS links the single-carbon folate pathway to the methionine cycle, and is a potentially important site for metabolic control. Nonetheless, there have been no prior reports of its regulation by extracellular signaling pathways. Our studies demonstrate the ability of IGF-1 and dopamine to increase MS activity via a mechanism that requires the activity of both PI3- kinase and MAP-kinase pathways. MS activity is a major determinant of both homocysteine and SAH levels, and the efficiency of methylation reactions is governed by the [SAM] to[SAH] ratio.12,13 These relationships imply that growth factors, by increasing PI3- and MAP-kinase activity, can facilitate transmethylation reactions, via activation of MS. Conversely, agents interfering with this mechanism will impair methylation. Our studies also provide evidence that ethanol, heavy metals and the vaccine preservative thimerosal potently interfere with MS activation and impair
folate-dependent methylation. Since each of these agents has been linked to developmental disorders, [??] our findings suggest that impaired methylation,
particularly impaired DNA methylation in response to growth factors, may be an important molecular mechanism leading to developmental disorders. DNA methylation is a crucial regulator of gene expression that has been linked to several developmental disorders. The majority of Rett syndrome cases are caused by MeCp2 mutations that interfere with MeCP2 to binding to methylated CpG sites in the genome.3 As a consequence, the protein complex necessary for histone modification and gene silencing fails to form, leading to dysregulated gene expression. Fragile-X syndrome is associated with localized hypermethylation of unstable CGG repeats at fragile sites on the X-chromosome (Xq27.3).4 Impaired MS activity could therefore contribute to developmental disorders via altered patterns of DNA methylation. Growth factors (eg nerve growth factor, brain derived neurotrophic factor and IGF-1) promote development of the neuronal phenotype and support the function and survival of differentiated nerves The capacity to activate simultaneously both PI3- kinase and MAP-kinase pathways is a feature of many growth factors.37–39 Blocking the methionine cycle (eg
with inhibitors of SAH hydrolase) interferes with neurotrophic responses,10,11 indicating an essential role for methylation in growth factor action. Since
differences in cellular phenotype reflect varied patterns of methylation-dependent gene silencing, it is reasonable to hypothesize that growth factors might directly or indirectly modulate genomic methylation status during development. IGF-1 exerts trophic and antiapoptotic effects on a wide variety of cell types, and its involvement in brain development is well documented.40,41 In addition to its neurotrophic action, IGF-1 promotes differentiation and survival of myelin-producing oligodendrocytes,42 an action in which divalent copper plays an integral role. Thus the chelation of copper causes demyelination and an upregulation of IGF-1.43 Vitamin B12 deficiency44 and chronic nitrous
oxide exposure,45 both of which impair MS, also cause demyelination. We found that Cu2þ promotes MS activity (Table 1) and protects against the inhibitory effects of other metals (Figure 5e), while Cu2þ chelation has an opposite effect (Figures 5a and f). Thus oligodendrocytes provide a specific example of how
IGF-1, metal ions and methylation can combine to affect cellular differentiation
and brain development. During postnatal development, myelination is critical for the specification of fixed connections between brain regions (ie hard-wiring), and there have been a number of reports of abnormal white matter (ie myelination) in autism.46–48 Neurodevelopmental insults affecting myelination could lead to
abnormal neural connections, resulting in the enhancement of certain relationships, but deficiencies in others, as is frequently observed in autism. Reduced IGF-1 levels have been reported in autism,49 which may also
contribute to impaired myelination. Fetal ethanol exposure, consequent to
maternal alcohol use, leads to the complex disorder known as fetal alcohol syndrome.5 In humans and animal models, IGF-1 levels are reduced after fetal
ethanol exposure, and the decrease is sustained through postnatal development.50,51 Ethanol increases homocysteine levels in animals and man,52,53 in association with impaired MS activity.54 Ethanol potently inhibits
basal- and IGF-1-stimulated MS activity (Table 1), reduces folate-dependent methylation (Figure 3a), and blocks the ability of IGF-1 to increase DNA methylation (Table 2). The IC50 for ethanol inhibition of methylation (8mM) corresponds to blood levels produced by only one or two drinks, indicating a
potential for adverse effects on methylation events from only moderate drinking. In a related finding, IGF-1 has been shown to promote recovery from carbon tetrachloride-induced cirrhosis, by increasing DNA methylation and normalizing gene expression.55 As illustrated in Figure 7, MS has two substrates, homocysteine and the dopamine D4 receptor in its homocysteine state. Dopamine-stimulated PLM, measured with [14C]formate, reflects D4 receptor-directed
MS activity and ethanol increases dopamine-stimulated PLM (Figure 4d), in contrast to its inhibition of homocysteine-directed MS activity (Table 1). These
results indicate that ethanol promotes the ability of MS to utilize the D4 receptor
as a substrate, while simultaneously decreasing homocysteine methylation. Dopamine increases MS activity and folate-dependent PLM in SH-SY5Y cells via a mechanism requiring both PI3-kinase and MAP-kinase activation (Table 1, Figures 3b and c), and this increase will promote the efficiency of the D4 receptor-mediated PLM cycle. Although the functional role of dopamine stimulated PLM is not fully understood, the incidence of ADHD is linked to genetic variations within the D4 receptor gene,18 and it has been proposed that dopamine-stimulated PLM plays a central role in attention.19 Impairment of MS could therefore adversely
affect the capacity for attention and could contribute to the risk of ADHD. Lead exposure, particularly early in life, causes growth retardation along with impairments in attention and cognitive development,6 and government guidelines establish blood concentrations exceeding 500nM as indicative of lead poisoning.56 An increase in blood lead levels from 1 to 10 mg/dl (120–1200 nM) is associated with an IQ decrease of 7.4 points.28 Since lead inhibits IGF-1-stimulated methylation with an IC50 value of 100nM (Figure 5c), impaired MS could contribute to developmental delay and impaired cognition associated with lead poisoning. Mercury exposure has been suggested as a possible cause of autism7 and methylmercury is a wellrecognized neurotoxin.57 A blood mercury level of
29nM has been recommended by the Environmental Protection Agency as a reference value for defining toxic exposure.58 We found that inorganic mercury
inhibits IGF-1-stimulated methylation with an IC50 of 15nM (Figure 5c). Aluminum salts are used as vaccine adjuvants, based on their ability to improve dendritic cell response to presented antigens. The aluminum content of vaccines varies from 0.125 to 0.85 mg/dose, which would produce concentrations of
approximately 0.7 to 4.5 mM, if uniformly distributed in the body water of a 7 kg infant. These concentrations produce greater than 50% inhibition of both IGF-1-
and dopamine-stimulated methylation, raising the possibility that aluminum concentrations produced by vaccination might adversely affect methylation
events. In light of the importance of MS in regulating DNA methylation59,60 and the central role of DNA methylation in development,9 we propose that metal
exposures, including lead, mercury and aluminum, may contribute to developmental syndromes via their inhibitory effects on signaling pathways that regulate MS activity. Thimerosal is an ethylmercury derivative of thiosalicylate,
widely used as a preservative to block the growth of contaminating organisms in biological products. It was included in most vaccines in the US until 1999, when the FDA initiated a precautionary program calling for ‘thimerosal-free’ vaccines. Most, but not all, vaccines are now ‘thimerosal-free’, meaning that they contain less than 0.5 mg thimerosal/dose.61 Thimerosal inhibits IGF-1 and
dopamine stimulated methylation with an IC50 near 1 nM, (Figures 5e and f), indicating its potential for disrupting normal growth factor control over methylation. Thiosalicylate itself also inhibited methylation,
presumably by chelating Cu2þ, but was more than 100-fold less potent than thimerosal (Figure 5f), indicating that the ethylmercury in thimerosal is
responsible for its inhibitory effect. The presence of added Cu2þ, however, significantly offsets thimerosal- induced inhibition, reflecting competition between
promotional and inhibitory effects of metals on the PI3-kinase MS pathway. Thus, the toxicity of thimerosal in the body may depend upon the concentrations of metal ions that provide either additive toxicity or protective effects on PI3-kinase
signaling. Thimerosal has been reported to activate apoptosis in lymphocytes62 and in cultured human cortical neurons,63 consistent with the inhibition of
the PI3-kinase signaling pathway. A single thimerosal-containing vaccination produces acute ethylmercury blood levels of 10– 30 nM,64 and blood samples in 2-month-old infants, obtained 3–20 days after vaccination, contain 3.8 20.6nM ethylmercury.65 Our studies therefore indicate the potential for thimerosal to cause adverse effects on MS activity at concentrations well below the levels produced by individual thimerosal-containing vaccines.
If impaired MS activity does indeed contribute to neurodevelopmental toxicity, limitations in other pathways that support homocysteine methylation could
predispose individuals to higher risk. Since SAH hydrolase is reversible, the concentration of adenosine determines the probability that homocysteine will be reconverted to SAH (Figure 7). Adenosine deaminase activity is reduced in autism,66 which would lead to higher adenosine levels and enhanced SAH formation. A polymorphism in the adenosine deaminase gene, that gives rise to a lower activity enzyme, is over-represented in autism.67,68 Adenosine is formed
by the action of 50-nucleotidase on AMP, and Page et al69 found eight- to 10-fold higher 50- nucleotidase activity in association with an ‘autismlike’ developmental disorder. Each of these autism associated metabolic abnormalities could synergize with reduced MS activity to impair methylation. Mutations in the adenylosuccinate lyase (ASL) gene are a rare but penetrant cause of autism.2 Lower enzyme activity blocks de novo purine synthesis in conjunction with a massive buildup of preblock intermediates that are ultimately excreted in the
urine. As illustrated in Figure 7, increased flux of folate-derived single-carbon groups to purine synthesis restricts the availability of 5-methylTHF for MS. Moreover, increased flux of single-carbon groups toward de novo purine synthesis is common in autism,70 as well as in Lesch–Nyhan Syndrome,71 and this may increase sensitivity to neurodevelopmental toxins acting on MS. A recent rise in autism incidence72 has triggered concerns that an environmental factor might be
promoting developmental disorders. Attention has been directed towards vaccines as a possible cause of the rise, since there has been a significant increase in
the number of required vaccinations since the early 1980s.7,28,29 Depending on infant weight and vaccination schedule, the vaccine-associated dosage of
ethylmercury during the initial 24 months of life approached or exceeded federal guidelines for methylmercury exposure.61 A recent analysis of data from the Vaccine Adverse Event Reporting System, maintained by the Centers for Disease
Control, found a significant correlation between the use of the thimerosal-containing formulation (vs the thimerosal- free formulation) of the Diphtheria, Tetanus and acellular Pertussis (DTaP) vaccine and autism.31 The discovery of the PI3-kinase/MAP-kinase/MS pathway, and its potent inhibition by developmental neurotoxins, including vaccine components thimerosal and aluminum, provides a potential molecular explanation for how increased use of vaccines could promote an increase in the incidence of autism. The increased
incidence of ADHD, which preceded the more recent rise in autism, could represent an alternative manifestation of vaccine-associated neurodevelopmental toxicity, since the D4 dopamine receptor is linked to ADHD18 and its PLM function depends on MS.15
There are important limitations to our findings. We utilized a transformed cell line, and molecular events in tumor-derived cells might not mirror those in normal cells. SH-SY5Ycells are undifferentiated neuronal precursor cells, so it remains unclear whether growth factors and/or dopamine modulate MS activity and DNA methylation in fully differentiated cells. On the other hand, undifferentiated cells may provide a particularly appropriate model system for the study of developmental disorders. It is obvious that biochemical studies under cultured cells conditions do not replicate the complex in vivo environment, in terms of ambient metal ion concentrations, redox conditions and other factors that could influence
methylation events. Further investigation of the in vivo and in vitro effects of heavy metals on growth factor-induced cellular differentiation is needed. While our studies focused exclusively on MS- and methylation-related events, we can speculate that other PI3-kinase signaling pathways may also be affected by metal ions. In summary, IGF-1 and dopamine activate methionine kinase in SH-SY5Y human neuroblastoma cells via a PI3-kinase and MAP-kinase-dependent mechanism, and the activation is associated with increased DNA methylation. Several neurodevelopmental toxins inhibit this newly recognized pathway with
remarkable potency, suggesting that their pathological effects might result from interruption of growth factor-initiated increases in DNA methylation and normal epigenetic regulation of gene expression. Further studies are needed to establish
the functional significance of regulated MS activity and to evaluate the possibility that vaccine components (ie thimerosal and aluminum) may have contributed to the risk of autism, ADHD and other developmental disorders.

Take away all of the possible mechanisms, the speculation and the what-ifs, we are left with a very short communication. Too short to be called brief. If we replace any of the highlighted "waffle words" with their opposites, the conclusions and discussions are no more or less accurate.

We found that insulin-like growth factor-1 (IGF-1)- and dopamine-stimulated
methionine synthase (MS) activity and folate-dependent methylation of
phospholipids in SH-SY5Y human neuroblastoma cells, via a PI3-kinase- and
MAP-kinase-dependent mechanism. The stimulation of this pathway increased DNA
methylation, while its inhibition increased methylation-sensitive gene
expression. Ethanol potently interfered with IGF-1 activation of MS and blocked
its effect on DNA methylation, whereas it did not inhibit the effects of
dopamine. Metal ions potently affected IGF-1 and dopamine-stimulated MS
activity, as well as folate-dependent phospholipid methylation: Cu(2+) promoted
enzyme activity and methylation, while Cu(+), Pb(2+), Hg(2+) and Al(3+) were
inhibitory. The ethylmercury-containing preservative thimerosal inhibited both
IGF-1- and dopamine-stimulated methylation with an IC(50) of 1 nM and eliminated
MS activity.

That's all folks. That's the important part right there. Ignore the rest at your own peril.