Thimerosal's ethylmercury: Aschner's summary versus Geier et al's summary

Teresa Binstock
Researcher in Developmental & Behavioral Neuroanatomy
December 12, 200
9


In a 2006 review about metallothioneins [MTs] and mercury (1), Michael Aschner includes a passage about ethylmercury (eHg; 2). Dr. Aschner's paragraph about eHg stands in stark contrast to an ethylmercury summary presented by Geier et al (3). A comparison of the two articles' sections about ethylmercury is instructive and prompts concern: Why, as a professor and researcher seemingly qualified to write intelligently about ethylmercury, did Michael Aschner write inaccurately and misleadingly about eHg?

First, consider Dr. Aschner's first sentence of the section entitled, "Ethylmercury and MTs". He wrote, "Studies have yet to be performed to address the effects of EtHg, a vaccine preservative containing 50% EtHg, on MTs in brain." Here's a minor quibble. That sentence states that EtHg is a vaccine preservative "containing 50% EtHg".  More accurate would have been to write that, Thimerosal is used as a vaccine preservative and is approximately 50% mercury by weight. 

More significantly, the rest of his paragraph about "Ethylmercury and MTs" conveys the impression of Not To Worry about thimerosal. Dr. Aschner wrote:
        "EtHg decomposes faster than MeHg. Because passage through the blood-brain barrier favors small molecules and MeHg is actively transported, Hg concentrations in the brain are higher following exposure to MeHg. Hg also clears faster after administration of EtHg versus MeHg. Because metabolic rates (basic metabolism, rates of loss from the body burden) are related to the fractional power of body weight (allometric relationship), Hg clears faster from infants, thus blood Hg concentrations for MeHg underestimate the safe exposure range for EtHg... These observations suggest, but have yet to be verified, that MT induction upon EtHg is most likely relatively small in comparisons to Hg0 and even inorganic mercury in the form of Hg2+." (1)

However, injected ethylmercury may not be as benign as Dr. Aschner's rhetoric suggests. Consider an ethylmercury summary by more thorough reviewers, who describe ethylmercury-brain findings in a primate model (21) published two years before Dr. Ashner's 2006 review:
        "Researchers evaluated infant monkeys following oral administration of methyl-Hg hydroxide or injected doses of thimerosal, sodium ethyl-Hg thiosalicylate, comparable to the dosing schedule (weight- and ageadjusted) that US children received during the 1990s... They determined that the maximum Hg content in the brains of the thimerosal-treated infant monkeys averaged about 40-50 parts-per-billion. In addition, they calculated that the half-life for organic Hg in the brain of the infant monkeys examined was about 14 days. By contrast, they determined that maximum Hg content in the brains of methyl-Hg treated infant monkeys averaged about 80-120 parts-per-billion. In this case, they calculated that the half-life for organic Hg in the brain of the infant monkeys examined was about 58 days. Based on these results, it was demonstrated that infant low-dose organic Hg exposure, mimicking the Hg exposure received by US children through the 1990s immunization schedule, was able to induce significant levels of Hg in the monkey brain, and that this organic Hg was present in the brain for several weeks postdosing.
       "In addition, post-dosing-schedule testing found the concentration of Hg2+ (formed from the ethyl-Hg entering the brain) averaged 16 parts-per-billion in the brains of the thimerosal-treated infant monkeys (all had a measurable level), whereas methyl-Hg treated infant monkeys had significantly less than half as much Hg2+ (almost half had a level below the method’s detection limit). Moreover, the half-life of Hg2+ in the monkeys’ brains was too long to estimate from the available data (no significant measurable decline was detectable by 120 days). Additionally, it was previously reported that, as a result of the significant Hg2+ fraction of Hg observed in the brain following injection of thimerosal, a longer biological time was observed for the Hg in the brain from ethyl-Hg than for the Hg from methyl-Hg..." (3)

Initial conclusion: After being injected by a physician or nurse, thimerosal's ethylmercury is not as benign as Dr. Aschner would have us believe if we rely upon his 2006 essay.

Furthermore, the levels of eHg present in healthy human infants after vaccination with a thimerosal-containing vaccine (4) are sufficiently high so as to alter methionine synthase (5). This finding has additional significance because Dr. Pichichero's sample size was small (4) and, by utilizing
healthyinfants, the study may not have included infants with increased susceptibility (eg, 6-7) and did not include infants who - at the time of deliberate thimerosal injection - were sick or had been recently sick. Importantly, thimerosal inhibits glutathione function (8).

A diligent reader of thimerosal literature might overlook one study's perfunctory and misleading summary about ethylmercury (eg, 1), but for Dr. Aschner, whitewashing adverse effects of thimerosal has been repeated (reviewed in 9).

Furthermore, despite news media summaries that tell us how safe physician- and nurse-injected thimerosal is, a growing number of findings indicate that thimerosal injections are associated with an increased need for special education services (10), with an increased rate of autism (11), with mitochondrial dysfunction (eg, 12-14). Moreover, the ethylmercury in thimerosal may interact additively, cumulatively, or synergistically with airborne mercury (eg, 15-17) and with other toxicants (eg, pesticides; 18-19).

Fortunately, a thorough review about thimerosal has been written by Geier and colleagues and is free online (20).


References:

1.
Metallothioneins: mercury species-specific induction and their potential role in attenuating neurotoxicity
Aschner M, Syversen T, Souza DO, Rocha JB.
Exp Biol Med (Maywood). 2006 Oct;231(9):1468-73.
{free online}
http://www.ebmonline.org/cgi/reprint/231/9/1468

2. EtHg is the traditional acronym for ethylmercury, but I prefer the shortened abbreviation, eHg, which is easily differentiated from mHg, which can be an abbreviation for methylmercury.

3.
A comprehensive review of mercury provoked autism
Geier DA, King PG, Sykes LK, Geier MR.
Indian J Med Res. 2008 Oct;128(4):383-411.
{free online}
http://www.icmr.nic.in/ijmr/2008/october/1004.pdf

4.
Mercury concentrations and metabolism in infants receiving vaccines containing thiomersal: a descriptive study
Pichichero ME, Cernichiari E, Lopreiato J, Treanor J.
Lancet. 2002 Nov 30;360(9347):1737-41.

BACKGROUND: Thiomersal is a preservative containing small amounts of ethylmercury that is used in routine vaccines for infants and children. The effect of vaccines containing thiomersal on concentrations of mercury in infants' blood has not been extensively assessed, and the metabolism of ethylmercury in infants is unknown. We aimed to measure concentrations of mercury in blood, urine, and stools of infants who received such vaccines. METHODS: 40 full-term infants aged 6 months and younger were given vaccines that contained thiomersal (diptheria-tetanus-acellular pertussis vaccine, hepatitis B vaccine, and in some children Haemophilus influenzae type b vaccine). 21 control infants received thiomersal-free vaccines. We obtained samples of blood, urine, and stools 3-28 days after vaccination. Total mercury (organic and inorganic) in the samples was measured by cold vapour atomic absorption. FINDINGS: Mean mercury doses in infants exposed to thiomersal were 45.6 microg (range 37.5-62.5) for 2-month-olds and 111.3 microg (range 87.5-175.0) for 6-month-olds. Blood mercury in thiomersal-exposed 2-month-olds ranged from less than 3.75 to 20.55 nmol/L (parts per billion); in 6-month-olds all values were lower than 7.50 nmol/L. Only one of 15 blood samples from controls contained quantifiable mercury. Concentrations of mercury were low in urine after vaccination but were high in stools of thiomersal-exposed 2-month-olds (mean 82 ng/g dry weight) and in 6-month-olds (mean 58 ng/g dry weight). Estimated blood half-life of ethylmercury was 7 days (95% CI 4-10 days). INTERPRETATION: Administration of vaccines containing thiomersal does not seem to raise blood concentrations of mercury above safe values in infants. Ethylmercury seems to be eliminated from blood rapidly via the stools after parenteral administration of thiomersal in vaccines.

5.
Activation of methionine synthase by insulin-like growth factor-1 and dopamine: a target for neurodevelopmental toxins and thimerosal
Waly M et al.
Mol Psychiatry. 2004 Apr;9(4):358-70.
{free online}
http://www.nature.com/mp/journal/v9/n4/pdf/4001476a.pdf

Methylation events play a critical role in the ability of growth factors to promote normal development. Neurodevelopmental toxins, such as ethanol and heavy metals, interrupt growth factor signaling, raising the possibility that they might exert adverse effects on methylation. 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. Our findings outline a novel growth factor signaling pathway that regulates MS activity and thereby modulates methylation reactions, including DNA methylation. The potent inhibition of this pathway by ethanol, lead, mercury, aluminum and thimerosal suggests that it may be an important target of neurodevelopmental toxins.

6.
Homozygous gene deletions of the glutathione S-transferases M1 and T1 are associated with thimerosal sensitization
Westphal GA et al.
Int Arch Occup Environ Health. 2000 Aug;73(6):384-8.

7.
Metabolic endophenotype and related genotypes are associated with oxidative stress in children with autism
James SJ et al.
Am J Med Genet B Neuropsychiatr Genet. 2006 Dec 5;141B(8):947-56.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2610366/pdf/nihms68264.pdf

Autism is a behaviorally defined neurodevelopmental disorder usually diagnosed in early childhood that is characterized by impairment in reciprocal communication and speech, repetitive behaviors, and social withdrawal. Although both genetic and environmental factors are thought to be involved, none have been reproducibly identified. The metabolic phenotype of an individual reflects the influence of endogenous and exogenous factors on genotype. As such, it provides a window through which the interactive impact of genes and environment may be viewed and relevant susceptibility factors identified. Although abnormal methionine metabolism has been associated with other neurologic disorders, these pathways and related polymorphisms have not been evaluated in autistic children. Plasma levels of metabolites in methionine transmethylation and transsulfuration pathways were measured in 80 autistic and 73 control children. In addition, common polymorphic variants known to modulate these metabolic pathways were evaluated in 360 autistic children and 205 controls. The metabolic results indicated that plasma methionine and the ratio of S-adenosylmethionine (SAM) to S-adenosylhomocysteine (SAH), an indicator of methylation capacity, were significantly decreased in the autistic children relative to age-matched controls. In addition, plasma levels of cysteine, glutathione, and the ratio of reduced to oxidized glutathione, an indication of antioxidant capacity and redox homeostasis, were significantly decreased. Differences in allele frequency and/or significant gene-gene interactions were found for relevant genes encoding the reduced folate carrier (RFC 80G > A), transcobalamin II (TCN2 776G > C), catechol-O-methyltransferase (COMT 472G > A), methylenetetrahydrofolate reductase (MTHFR 677C > T and 1298A > C), and glutathione-S-transferase (GST M1). We propose that an increased vulnerability to oxidative stress (endogenous or environmental) may contribute to the development and clinical manifestations of autism.

8.
Inhibition of the human erythrocytic glutathione-S-transferase T1 (GST T1) by thimerosal
Muller M et al.
Int J Hyg Environ Health. 2001 Jul;203(5-6):479-81.

9.
Michael Aschner & Sandra Ceccatelli: Sins of Omission Regarding Thimerosal
Teresa Binstock
here

10.
Hepatitis B triple series vaccine and developmental disability in US children aged 1-9 years
 Gallagher C, Goodman M. Toxicol Environ Chem 2008 90(5):997-1008.
{free online}
http://fourteenstudies.org/pdf/hep_b.pdf

This study investigated the association between vaccination with the Hepatitis B triple series vaccine prior to 2000 and developmental disability in children aged 1-9 years (n = 1824), proxied by parental report that their child receives early intervention or special education services (EIS). National Health and Nutrition Examination Survey 1999-2000 data were analyzed and adjusted for survey design by Taylor Linearization using SAS version 9.1 software, with SAS callable SUDAAN version 9.0.1.
The odds of receiving EIS were approximately nine times as great for vaccinated boys (n = 46) as for unvaccinated boys (n = 7), after adjustment for confounders. This study found statistically significant evidence to suggest that boys in United States who were vaccinated with the triple series Hepatitis B vaccine, during the time period in which vaccines were manufactured with thimerosal, were more susceptible to developmental disability than were unvaccinated boys.

11.
Hepatitis B vaccination of male neonates and autism
[conference abstract as published]
CM Gallagher, MS Goodman, Graduate Program in Public
Health, Stony Brook University Medical Center, Stony Brook, NY
Annals of Epidemiology, p659
Vol. 19, No. 9 Abstracts (ACE) September 2009: 651–680

PURPOSE: Universal newborn immunization with hepatitis B vaccine was recommended in 1991; however, safety findings are mixed. The Vaccine Safety Datalink Workgroup reported no association between hepatitis B vaccination at birth and febrile episodes or neurological adverse events. Other studies found positive associations between hepatitis B vaccination and ear infection, pharyngitis, and chronic arthritis; as well as receipt of early intervention/special education services (EIS); in probability samples of
U.S. children. Children with autistic spectrum disorder (ASD) comprise a growing caseload for EIS. We evaluated the association between hepatitis B vaccination of male neonates and parental report of ASD.
METHODS: This cross-sectional study used U.S. probability samples obtained from National Health Interview Survey 1997–2002 datasets. Logistic regression modeling was used to estimate the effect of neonatal hepatitis B vaccination on ASDrisk amongboys age 3–17 years with shot records, adjusted for race, maternal education, and two-parent household.
RESULTS:Boyswho received the hepatitis B vaccine during the first month of life had 2.94 greater odds for ASD (nZ31 of 7,486; OR Z 2.94; p Z 0.03; 95% CI Z 1.10, 7.90) compared to later- or unvaccinated boys. Non-Hispanicwhite boys were 61% less likely to have ASD(ORZ0.39; pZ0.04; 95% CIZ0.16, 0.94) relative to non-white boys.
CONCLUSION: Findings suggest that U.S. male neonates vaccinated with hepatitis B vaccine had a 3-fold greater risk of ASD; risk was greatest for non-white boys.

12.
Biochemical and molecular basis of thimerosal-induced apoptosis in T cells: a major role of mitochondrial pathway
Makani S et al.
Division of Basic and Clinical Immunology, University of California
Genes Immun. 2002 Aug;3(5):270-8.
http://www.nature.com/gene/journal/v3/n5/abs/6363854a.html

The major source of thimerosal (ethyl mercury thiosalicylate) exposure is childhood vaccines. It is believed that the children are exposed to significant accumulative dosage of thimerosal during the first 2 years of life via immunization. Because of health-related concerns for exposure to mercury, we examined the effects of thimerosal on the biochemical and molecular steps of mitochondrial pathway of apoptosis in Jurkat T cells. Thimerosal and not thiosalcylic acid (non-mercury component of thimerosal), in a concentration-dependent manner, induced apoptosis in T cells as determined by TUNEL and propidium iodide assays, suggesting a role of mercury in T cell apoptosis. Apoptosis was associated with depolarization of mitochondrial membrane, release of cytochrome c and apoptosis inducing factor (AIF) from the mitochondria, and activation of caspase-9 and caspase-3, but not of caspase-8. In addition, thimerosal in a concentration-dependent manner inhibited the expression of XIAP, cIAP-1 but did not influence cIAP-2 expression. Furthermore, thimerosal enhanced intracellular reactive oxygen species and reduced intracellular glutathione (GSH). Finally, exogenous glutathione protected T cells from thimerosal-induced apoptosis by upregulation of XIAP and cIAP1 and by inhibiting activation of both caspase-9 and caspase-3. These data suggest that thimerosal induces apoptosis in T cells via mitochondrial pathway by inducing oxidative stress and depletion of GSH.

13.
Mitochondrial mediated thimerosal-induced apoptosis in a human neuroblastoma cell line (SK-N-SH)
Humphrey ML, Cole MP, Pendergrass JC, Kiningham KK.
Neurotoxicology. 2005 Jun;26(3):407-16.

Environmental exposure to mercurials continues to be a public health issue due to their deleterious effects on immune, renal and neurological function. Recently the safety of thimerosal, an ethyl mercury-containing preservative used in vaccines, has been questioned due to exposure of infants during immunization. Mercurials have been reported to cause apoptosis in cultured neurons; however, the signaling pathways resulting in cell death have not been well characterized. Therefore, the objective of this study was to identify the mode of cell death in an in vitro model of thimerosal-induced neurotoxicity, and more specifically, to elucidate signaling pathways which might serve as pharmacological targets. Within 2 h of thimerosal exposure (5 microM) to the human neuroblastoma cell line, SK-N-SH, morphological changes, including membrane alterations and cell shrinkage, were observed. Cell viability, assessed by measurement of lactate dehydrogenase (LDH) activity in the medium, as well as the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, showed a time- and concentration-dependent decrease in cell survival upon thimerosal exposure. In cells treated for 24 h with thimerosal, fluorescence microscopy indicated cells undergoing both apoptosis and oncosis/necrosis. To identify the apoptotic pathway associated with thimerosal-mediated cell death, we first evaluated the mitochondrial cascade, as both inorganic and organic mercurials have been reported to accumulate in the organelle. Cytochrome c was shown to leak from the mitochondria, followed by caspase 9 cleavage within 8 h of treatment. In addition, poly(ADP-ribose) polymerase (PARP) was cleaved to form a 85 kDa fragment following maximal caspase 3 activation at 24 h. Taken together these findings suggest deleterious effects on the cytoarchitecture by thimerosal and initiation of mitochondrial-mediated apoptosis.

14.
Thimerosal induces neuronal cell apoptosis by causing cytochrome c and apoptosis-inducing factor release from mitochondria
Yel L et al.
Int J Mol Med. 2005 Dec;16(6):971-7.

There is a worldwide increasing concern over the neurological risks of thimerosal (ethylmercury thiosalicylate) which is an organic mercury compound that is commonly used as an antimicrobial preservative. In this study, we show that thimerosal, at nanomolar concentrations, induces neuronal cell death through the mitochondrial pathway. Thimerosal, in a concentration- and time-dependent manner, decreased cell viability as assessed by calcein-ethidium staining and caused apoptosis detected by Hoechst 33258 dye. Thimerosal-induced apoptosis was associated with depolarization of mitochondrial membrane, generation of reactive oxygen species, and release of cytochrome c and apoptosis-inducing factor (AIF) from mitochondria to cytosol. Although thimerosal did not affect cellular expression of Bax at the protein level, we observed translocation of Bax from cytosol to mitochondria. Finally, caspase-9 and caspase-3 were activated in the absence of caspase-8 activation. Our data suggest that thimerosal causes apoptosis in neuroblastoma cells by changing the mitochondrial microenvironment.

15.
Environmental mercury release, special education rates, and autism disorder: an ecological study of Texas
Palmer RF et al.
Health Place. 2006 Jun;12(2):203-9.

16.
Autism spectrum disorders in relation to distribution of hazardous air pollutants in the san francisco bay area
Windham GC et al.
Environ Health Perspect. 2006 Sep;114(9):1438-44.

17.
Proximity to point sources of environmental mercury release as a predictor of autism prevalence
Palmer RF et al.
Health Place. 2009 Mar;15(1):18-24.

18.
Paraoxonase [PON1] gene variants are associated with autism in North America, but not in Italy: possible regional specificity in gene-environment interactions
 D'Amelio et al.
Mol Psychiatry. 2005 Nov;10(11):1006-16.

Organophosphates (OPs) are routinely used as pesticides in agriculture and as insecticides within the household. Our prior work on Reelin and APOE delineated a gene-environment interactive model of autism pathogenesis, whereby genetically vulnerable individuals prenatally exposed to OPs during critical periods in neurodevelopment could undergo altered neuronal migration, resulting in an autistic syndrome. Since household use of OPs is far greater in the USA than in Italy, this model was predicted to hold validity in North America, but not in Europe. Here, we indirectly test this hypothesis by assessing linkage/association between autism and variants of the paraoxonase gene (PON1) encoding paraoxonase, the enzyme responsible for OP detoxification. Three functional single nucleotide polymorphisms, PON1 C-108T, L55M, and Q192R, were assessed in 177 Italian and 107 Caucasian-American complete trios with primary autistic probands. As predicted, Caucasian-American and not Italian families display a significant association between autism and PON1 variants less active in vitro on the OP diazinon (R192), according to case-control contrasts (Q192R: chi2=6.33, 1 df, P<0.025), transmission/disequilibrium tests (Q192R: TDT chi2=5.26, 1 df, P<0.025), family-based association tests (Q192R and L55M: FBAT Z=2.291 and 2.435 respectively, P<0.025), and haplotype-based association tests (L55/R192: HBAT Z=2.430, P<0.025). These results are consistent with our model and provide further support for the hypothesis that concurrent genetic vulnerability and environmental OP exposure may possibly contribute to autism pathogenesis in a sizable subgroup of North American individuals.
 
19.
Maternal residence near agricultural pesticide applications and autism spectrum disorders among children in the California Central Valley
Roberts EM et al.
Environ Health Perspect. 2007 Oct;115(10):1482-9.

20. 
A review of Thimerosal (Merthiolate) and its ethylmercury breakdown product: specific historical considerations regarding safety and effectiveness
Geier DA, Sykes LK, Geier MR.
J Toxicol Environ Health B Crit Rev. 2007 Dec;10(8):575-96.
http://www.informaworld.com/smpp/ftinterface~content=a787584924~fulltext=713240930

21. Comparison of blood and brain mercury levels in infant monkeys exposed to methylmercury or vaccines containing thimerosal
Burbacher TM... Clarkson T.
Environ Health Perspect. 2005 Aug;113(8):1015-21.
{free online}
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1280342/pdf/ehp0113-001015.pdf

Thimerosal is a preservative that has been used in manufacturing vaccines since the 1930s. Reports have indicated that infants can receive ethylmercury (in the form of thimerosal) at or above the U.S. Environmental Protection Agency guidelines for methylmercury exposure, depending on the exact vaccinations, schedule, and size of the infant. In this study we compared the systemic disposition and brain distribution of total and inorganic mercury in infant monkeys after thimerosal exposure with those exposed to MeHg. Monkeys were exposed to MeHg (via oral gavage) or vaccines containing thimerosal (via intramuscular injection) at birth and 1, 2, and 3 weeks of age. Total blood Hg levels were determined 2, 4, and 7 days after each exposure. Total and inorganic brain Hg levels were assessed 2, 4, 7, or 28 days after the last exposure. The initial and terminal half-life of Hg in blood after thimerosal exposure was 2.1 and 8.6 days, respectively, which are significantly shorter than the elimination half-life of Hg after MeHg exposure at 21.5 days. Brain concentrations of total Hg were significantly lower by approximately 3-fold for the thimerosal-exposed monkeys when compared with the MeHg infants, whereas the average brain-to-blood concentration ratio was slightly higher for the thimerosal-exposed monkeys (3.5 +/- 0.5 vs. 2.5 +/- 0.3). A higher percentage of the total Hg in the brain was in the form of inorganic Hg for the thimerosal-exposed monkeys (34% vs. 7%). The results indicate that MeHg is not a suitable reference for risk assessment from exposure to thimerosal-derived Hg. Knowledge of the toxicokinetics and developmental toxicity of thimerosal is needed to afford a meaningful assessment of the developmental effects of thimerosal-containing vaccines.


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