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Evidence for Thimerosal Risk - Page 1


Neurochem Res. 2011 Feb 25. [Epub ahead of print]

Integrating Experimental (In Vitro and In Vivo) Neurotoxicity Studies of Low-dose Thimerosal Relevant to Vaccines.

Dórea JG.

Faculty of Health Sciences, Universidade de Brasília, C.P. 04322, 70919-970, Brasília, DF, Brazil, dorea@rudah.com.br.

Abstract

There is a need to interpret neurotoxic studies to help deal with uncertainties surrounding pregnant mothers, newborns and young children who must receive repeated doses of Thimerosal-containing vaccines (TCVs). This review integrates information derived from emerging experimental studies (in vitro and in vivo) of low-dose Thimerosal (sodium ethyl mercury thiosalicylate). Major databases (PubMed and Web-of-science) were searched for in vitro and in vivo experimental studies that addressed the effects of low-dose Thimerosal (or ethylmercury) on neural tissues and animal behaviour. Information extracted from studies indicates that: (a) activity of low doses of Thimerosal against isolated human and animal brain cells was found in all studies and is consistent with Hg neurotoxicity; (b) the neurotoxic effect of ethylmercury has not been studied with co-occurring adjuvant-Al in TCVs; (c) animal studies have shown that exposure to Thimerosal-Hg can lead to accumulation of inorganic Hg in brain, and that (d) doses relevant to TCV exposure possess the potential to affect human neuro-development. Thimerosal at concentrations relevant for infants' exposure (in vaccines) is toxic to cultured human-brain cells and to laboratory animals. The persisting use of TCV (in developing countries) is counterintuitive to global efforts to lower Hg exposure and to ban Hg in medical products; its continued use in TCV requires evaluation of a sufficiently nontoxic level of ethylmercury compatible with repeated exposure (co-occurring with adjuvant-Al) during early life.

PMID: 21350943 [PubMed - as supplied by publisher]

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Middle East Current Psychiatry
January 2011 - Volume 18 - Issue 1 - p 6–10
doi: 10.1097/01.XME.0000392842.64112.64
Study of some biomarkers in hair of children with autism
Elsheshtawy, Emana; Tobar, Salwaa; Sherra, Khalida; Atallah, Sohaylab; Elkasaby, Rashac
Correspondence to Eman Elsheshtawy, Department of Psychiatry Mansoura University Hospitals, Faculty of Medicine, Mansoura, Egypt Tel: +0187455403; fax: +26824738; e-mail: emanmdy@yahoo.com
Received August 8, 2010
Accepted November 25, 2010
Abstract
Introduction: Autism is a severe developmental disorder, which involves social withdrawal, communication deficits, and stereotypic repetitive behavior. The possible etiologies that precipitate autism symptoms remain controversial in many cases, but both genetic and environmental factors have been implicated. Mercury has gained much attention for a considerable period of time before other exacerbating or protective factors were suggested. The aim of this study was to investigate the relationship between autism and the level of some metals (namely mercury, lead, and copper) or zinc as a counteracting antioxidant element.
Methods: The study recruited 32 autistic children and 32 normal controls and all of them were subjected to KID-SCID, Childhood Autism Rating Scale (CARS), Stanford Binet intelligence test, and biochemical analysis of hair samples for the level of mercury, copper, lead and zinc.
Results: There were highly significant differences between the level of these substances in the hair of children with autism compared with controls, positive correlation of CARS score with both mercury and copper, while intelligence quotient has significant negative correlation with the level of lead in the hair. The level of zinc does not correlate with either CARS score or intelligence quotient.
Conclusion: These preliminary results suggest a complementary role for the studied elements in the pathogenesis of autistic disorder, which should be considered in the management plane.
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Folia Neuropathol. 2010;48(4):258-69.

Lasting neuropathological changes in rat brain after intermittent neonatal administration of thimerosal.

Olczak M, Duszczyk M, Mierzejewski P, Wierzba-Bobrowicz T, Majewska MD.

Department of Pharmacology and Physiology of the Nervous System, Institute of Psychiatry and Neurology, ul. Sobieskiego 9, Warsaw, Poland.

Abstract

Thimerosal, an organomercurial added as a preservative to some vaccines, is a suspected iatrogenic factor, possibly contributing to paediatric neurodevelopmental disorders including autism. We examined the effects of early postnatal administration of thimerosal (four i.m. injections, 12 or 240 μg THIM-Hg/kg, on postnatal days 7, 9, 11 and 15) on brain pathology in Wistar rats. Numerous neuropathological changes were observed in young adult rats which were treated postnatally with thimerosal. They included: ischaemic degeneration of neurons and "dark" neurons in the prefrontal and temporal cortex, the hippocampus and the cerebellum, pathological changes of the blood vessels in the temporal cortex, diminished synaptophysin reaction in the hippocampus, atrophy of astroglia in the hippocampus and cerebellum, and positive caspase-3 reaction in Bergmann astroglia. These findings document neurotoxic effects of thimerosal, at doses equivalent to those used in infant vaccines or higher, in developing rat brain, suggesting likely involvement of this mercurial in neurodevelopmental disorders.

PMID: 21225508 [PubMed - in process]Free Article

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Int J Immunopathol Pharmacol. 2010 Oct-Dec;23(4):1015-20.

Luteolin and thiosalicylate inhibit HgCl2 and thimerosal-induced VEGF release from human mast cells.

Asadi S, Zhang B, Weng Z, Angelidou A, Kempuraj D, Alysandratos KD, Theoharides TC.

Molecular Immunopharmacology and Drug Discovery Laboratory, Department of Pharmacology and Experimental Therapeutics, Tufts University School of Medicine and Tufts Medical Center, Boston.

Abstract

HgCl2 is a known environmental neurotoxin, but is also used as preservative in vaccines as thimerosal containing ethyl mercury covalently linked to thiosalicylate. We recently reported that mercury choloride (HgCl2) can stimulate human mast cells to release vascular endothelial growth factor (VEGF), which is also vasoactive and pro-inflammatory. Here we show that thimerosal induces significant VEGF release from human leukemic cultured LAD2 mast cells (at 1 microM 32612 pg/106 cells and 335.5±12 pg/106 cells at 10 microM) compared to control cells (242±21 pg/106 cells, n=5, p less than 0.05); this effect is weaker than that induced by HgCl2 at 10 microM (448±14 pg/106 cells) (n=3, p less than 0.05). In view of this finding, we hypothesize that the thiosalicylate component of thimerosal may have an inhibitory effect on VEGF release. Thimerosal (10 microM) added together with the peptide Substance P (SP) at 2 microM, used as a positive control, reduced VEGF release by 90 percent. Methyl thiosalicylate (1 or 10 microM) added with either SP or HgCl2 (10 microM) inhibited VEGF release by 100 percent, while sodium salicylate or ibuprofen had no effect. Pretreatment for 10 min with the flavonoid luteolin (0.1 mM) before HgCl2 or thimerosal compeletly blocked their effect. Luteolin and methyl thiosalicylate may be useful in preventing mercury-induced toxicity.

PMID: 21244751 [PubMed - in process] ______________________________________________________________________________

Acta Neurobiol Exp (Wars). 2010;70(2):147-64.

Influence of pediatric vaccines on amygdala growth and opioid ligand binding in rhesus macaque infants: A pilot study.

Hewitson L, Lopresti BJ, Stott C, Mason NS, Tomko J.

Department of Obstetrics and Gynecology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA. lch1@pitt.edu

Abstract

This longitudinal, case-control pilot study examined amygdala growth in rhesus macaque infants receiving the complete US childhood vaccine schedule (1994-1999). Longitudinal structural and functional neuroimaging was undertaken to examine central effects of the vaccine regimen on the developing brain. Vaccine-exposed and saline-injected control infants underwent MRI and PET imaging at approximately 4 and 6 months of age, representing two specific timeframes within the vaccination schedule. Volumetric analyses showed that exposed animals did not undergo the maturational changes over time in amygdala volume that was observed in unexposed animals. After controlling for left amygdala volume, the binding of the opioid antagonist [(11)C]diprenorphine (DPN) in exposed animals remained relatively constant over time, compared with unexposed animals, in which a significant decrease in [(11)C]DPN binding occurred. These results suggest that maturational changes in amygdala volume and the binding capacity of [(11)C]DPN in the amygdala was significantly altered in infant macaques receiving the vaccine schedule. The macaque infant is a relevant animal model in which to investigate specific environmental exposures and structural/functional neuroimaging during neurodevelopment.

PMID: 20628439 [PubMed - indexed for MEDLINE]Free Article

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Arch Toxicol. 2010 Nov;84(11):891-6. Epub 2010 Apr 13.

Identification and distribution of mercury species in rat tissues following administration of thimerosal or methylmercury.

Rodrigues JL, Serpeloni JM, Batista BL, Souza SS, Barbosa F Jr.

Faculdade de Ciências Farmacêuticas de Ribeirão Preto--FCFRP, Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, USP, Avenida do Café, s/n, Monte Alegre, Ribeirão Preto, SP 14040-903, Brazil.

Abstract

Methylmercury (Met-Hg) is one the most toxic forms of Hg, with a considerable range of harmful effects on humans. Sodium ethyl mercury thiosalicylate, thimerosal (TM) is an ethylmercury (Et-Hg)-containing preservative that has been used in manufacturing vaccines in many countries. Whereas the behavior of Met-Hg in humans is relatively well known, that of ethylmercury (Et-Hg) is poorly understood. The present study describes the distribution of mercury as (-methyl, -ethyl and inorganic mercury) in rat tissues (brain, heart, kidney and liver) and blood following administration of TM or Met-Hg. Animals received one dose/day of Met-Hg or TM by gavage (0.5 mg Hg/kg). Blood samples were collected after 6, 12, 24, 48, 96 and 120 h of exposure. After 5 days, the animals were killed, and their tissues were collected. Total blood mercury (THg) levels were determined by ICP-MS, and methylmercury (Met-Hg), ethylmercury (Et-Hg) and inorganic mercury (Ino-Hg) levels were determined by speciation analysis with LC-ICP-MS. Mercury remains longer in the blood of rats treated with Met-Hg compared to that of TM-exposed rats. Moreover, after 48 h of the TM treatment, most of the Hg found in blood was inorganic. Of the total mercury found in the brain after TM exposure, 63% was in the form of Ino-Hg, with 13.5% as Et-Hg and 23.7% as Met-Hg. In general, mercury in tissues and blood following TM treatment was predominantly found as Ino-Hg, but a considerable amount of Et-Hg was also found in the liver and brain. Taken together, our data demonstrated that the toxicokinetics of TM is completely different from that of Met-Hg. Thus, Met-Hg is not an appropriate reference for assessing the risk from exposure to TM-derived Hg. It also adds new data for further studies in the evaluation of TM toxicity.

PMID: 20386881 [PubMed - indexed for MEDLINE]

In other words, it is not settled….

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Cell Biol Toxicol. 2010 Apr;26(2):143-52. Epub 2009 Apr 9.

Induction of metallothionein in mouse cerebellum and cerebrum with low-dose thimerosal injection.

Minami T, Miyata E, Sakamoto Y, Yamazaki H, Ichida S.

Department of Life Sciences, Kinki University, Higashi-osaka, Osaka, Japan. minamita@life.kindai.ac.jp

Abstract

Thimerosal, an ethyl mercury compound, is used worldwide as a vaccine preservative. We previously observed that the mercury concentration in mouse brains did not increase with the clinical dose of thimerosal injection, but the concentration increased in the brain after the injection of thimerosal with lipopolysaccharide, even if a low dose of thimerosal was administered. Thimerosal may penetrate the brain, but is undetectable when a clinical dose of thimerosal is injected; therefore, the induction of metallothionein (MT) messenger RNA (mRNA) and protein was observed in the cerebellum and cerebrum of mice after thimerosal injection, as MT is an inducible protein. MT-1 mRNA was expressed at 6 and 9 h in both the cerebrum and cerebellum, but MT-1 mRNA expression in the cerebellum was three times higher than that in the cerebrum after the injection of 12 microg/kg thimerosal. MT-2 mRNA was not expressed until 24 h in both organs. MT-3 mRNA was expressed in the cerebellum from 6 to 15 h after the injection, but not in the cerebrum until 24 h. MT-1 and MT-3 mRNAs were expressed in the cerebellum in a dose-dependent manner. Furthermore, MT-1 protein was detected from 6 to 72 h in the cerebellum after 12 microg/kg of thimerosal was injected and peaked at 10 h. MT-2 was detected in the cerebellum only at 10 h. In the cerebrum, little MT-1 protein was detected at 10 and 24 h, and there were no peaks of MT-2 protein in the cerebrum. In conclusion, MT-1 and MT-3 mRNAs but not MT-2 mRNA are easily expressed in the cerebellum rather than in the cerebrum by the injection of low-dose thimerosal. It is thought that the cerebellum is a sensitive organ against thimerosal. As a result of the present findings, in combination with the brain pathology observed in patients diagnosed with autism, the present study helps to support the possible biological plausibility for how low-dose exposure to mercury from thimerosal-containing vaccines may be associated with autism.

PMID: 19357975 [PubMed - indexed for MEDLINE]

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Toxicol Appl Pharmacol. 2010 Mar 15;243(3):283-91. Epub 2009 Sep 2.

Mercury toxicokinetics--dependency on strain and gender.

Ekstrand J, Nielsen JB, Havarinasab S, Zalups RK, Söderkvist P, Hultman P.

Molecular and Immunological Pathology, Department of Clinical and Experimental Medicine, Linköping University, SE-58185 Linköping, Sweden.

Abstract

Mercury (Hg) exposure from dental amalgam fillings and thimerosal in vaccines is not a major health hazard, but adverse health effects cannot be ruled out in a small and more susceptible part of the exposed population. Individual differences in toxicokinetics may explain susceptibility to mercury. Inbred, H-2-congenic A.SW and B10.S mice and their F1- and F2-hybrids were given HgCl2 with 2.0 mg Hg/L drinking water and traces of (203)Hg. Whole-body retention (WBR) was monitored until steady state after 5 weeks, when the organ Hg content was assessed. Despite similar Hg intake, A.SW males attained a 20-30% significantly higher WBR and 2- to 5-fold higher total renal Hg retention/concentration than A.SW females and B10.S mice. A selective renal Hg accumulation but of lower magnitude was seen also in B10.S males compared with females. Differences in WBR and organ Hg accumulation are therefore regulated by non-H-2 genes and gender. Lymph nodes lacked the strain- and gender-dependent Hg accumulation profile of kidney, liver and spleen. After 15 days without Hg A.SW mice showed a 4-fold higher WBR and liver Hg concentration, but 11-fold higher renal Hg concentration, showing the key role for the kidneys in explaining the slower Hg elimination in A.SW mice. The trait causing higher mercury accumulation was not dominantly inherited in the F1 hybrids. F2 mice showed a large inter-individual variation in Hg accumulation, showing that multiple genetic factors influence the Hg toxicokinetics in the mouse. The genetically heterogeneous human population may therefore show a large variation in mercury toxicokinetics.

2010 Elsevier Inc. All rights reserved.

PMID: 19732784 [PubMed - indexed for MEDLINE]

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Clin Chim Acta. 2010 Nov 11;411(21-22):1580-6. Epub 2010 Jul 16.

Making sense of epidemiological studies of young children exposed to thimerosal in vaccines.

Dórea JG.

C.P. 04322, Universidade de Brasilia, 70919-970 Brasilia, DF, Brazil. dorea@rudah.com.br

Abstract

OBJECTIVE: To compare epidemiological studies dealing with neurological issues (compatible with Hg toxicity) linked to exposing newborns and infants to intramuscular doses of preservative-Hg resulting from vaccination with thimerosal-containing vaccines (TCV).

METHODS: Major databases were searched for studies that addressed neurodevelopment outcomes other than autism. Eight studies were identified and compared.

RESULTS: Information extracted from the studies done in the USA, the UK, and Italy is important in understanding the complex interplay of variables but insufficient to establish non-toxicity for infants and young children still receiving TCV: a) there is ambiguity in some studies reporting neurodevelopment outcomes that seem to depend on confounding variables; b) the risk of neurotoxicity due to low doses of thimerosal is plausible at least for susceptible infants; c) there is a need to address these issues in less developed countries still using TCV in pregnant mothers, newborns, and young children.

CONCLUSIONS: Since the use of TCV is still inevitable in many countries, this increases the need to protect vulnerable infants and promote actions that strengthen neurodevelopment. Developing countries should intensify campaigns that include breastfeeding among efforts to help prime the central nervous system to tolerate exposure to neurotoxic substances, especially thimerosal-Hg.

2010 Elsevier B.V. All rights reserved.

PMID: 20638374 [PubMed - indexed for MEDLINE]

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Acta Neurobiol Exp (Wars). 2010;70(2):196-208.

Age-dependent lower or higher levels of hair mercury in autistic children than in healthy controls.

Majewska MD, Urbanowicz E, Rok-Bujko P, Namyslowska I, Mierzejewski P.

Department of Pharmacology and Physiology of the Nervous System, Institute of Psychiatry and Neurology, Warsaw, Poland. majewska@ipin.edu.pl

Abstract

An association between autism and early life exposure to mercury is a hotly debated issue. In this study, 91 autistic Polish children, male and female, 3-4 and 7-9 years old, were compared to 75 age- and sex-matched healthy children with respect to: demographic, perinatal, clinical and developmental measures, parental age, birth order, morphometric measures, vaccination history, and hair mercury content. In demographic and perinatal measures there were no consistent differences between the autistic and control groups. Autistic children had a significantly greater prevalence of adverse reactions after vaccinations and abnormal development than controls. Between 45 and 80% of autistic children experienced developmental regress. Autistic children significantly differed from healthy peers in the concentrations of mercury in hair: younger autistics had lower levels, while older - higher levels than their respective controls. The results suggest that autistic children differ from healthy children in metabolism of mercury, which seems to change with age.

PMID: 20628443 [PubMed - indexed for MEDLINE]Free Article

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Talanta. 2010 Jan 15;80(3):1158-63.

Methylmercury and inorganic mercury determination in blood by using liquid chromatography with inductively coupled plasma mass spectrometry and a fast sample preparation procedure.

Rodrigues JL, de Souza SS, de Oliveira Souza VC, Barbosa F Jr.

Laboratório de Toxicologia e Essencialidade de Metais, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil.

Abstract

Despite the necessity to differentiate chemical species of mercury in clinical specimens, there are a limited number of methods for this purpose. Then, this paper describes a simple method for the determination of methylmercury and inorganic mercury in blood by using liquid chromatography with inductively coupled mass spectrometry (LC-ICP-MS) and a fast sample preparation procedure. Prior to analysis, blood (250microL) is accurately weighed into 15-mL conical tubes. Then, an extractant solution containing mercaptoethanol, l-cysteine and HCl was added to the samples following sonication for 15min. Quantitative mercury extraction was achieved with the proposed procedure. Separation of mercury species was accomplished in less than 5min on a C18 reverse-phase column with a mobile phase containing 0.05% (v/v) mercaptoethanol, 0.4% (m/v) l-cysteine, 0.06molL(-1) ammonium acetate and 5% (v/v) methanol. The method detection limits were found to be 0.25microgL(-1) and 0.1microgL(-1) for inorganic mercury and methylmercury, respectively. Method accuracy is traceable to Standard Reference Material (SRM) 966 Toxic Metals in Bovine Blood from the National Institute of Standards and Technology (NIST). The proposed method was also applied to the speciation of mercury in blood samples collected from fish-eating communities and from rats exposed to thimerosal. With the proposed method there is a considerable reduction of the time of sample preparation prior to speciation of Hg by LC-ICP-MS. Finally, after the application of the proposed method, we demonstrated an interesting in vivo ethylmercury conversion to inorganic mercury.

PMID: 20006068 [PubMed - indexed for MEDLINE]

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Neurotox Res. 2010 Jul;18(1):59-68. Epub 2009 Sep 16.

Are neuropathological conditions relevant to ethylmercury exposure?

Aschner M, Ceccatelli S.

Department of Pediatrics, Vanderbilt University Medical Center, Nashville, TN 37232, USA. michael.aschner@vanderbilt.edu

Abstract

Mercury and mercurial compounds are among the environmentally ubiquitous substances most toxic to both wildlife and humans. Once released into the environment from both natural and anthropogenic sources, mercury exists mainly as three different molecular species: elemental, inorganic, and organic. Potential health risks have been reported from exposure to all forms; however, of particular concern for human exposure relate to the potent neurotoxic effects of methylmercury (MeHg), especially for the developing nervous system. The general population is primarily exposed to MeHg by seafood consumption. In addition, some pharmaceuticals, including vaccines, have been, and some continue to be, a ubiquitous source of exposure to mercurials. A significant controversy has been whether the vaccine preservative ethylmercury thiosalicylate, commonly known as thimerosal, could cause the development of autism. In this review, we have discussed the hypothesis that exposure to thimerosal during childhood may be a primary cause of autism. The conclusion is that there are no reliable data indicating that administration of vaccines containing thimerosal is a primary cause of autism. However, one cannot rule out the possibility that the individual gene profile and/or gene-environment interactions may play a role in modulating the response to acquired risk by modifying the individual susceptibility.

PMID: 19756911 [PubMed - indexed for MEDLINE]

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J Environ Sci (China). 2009;21(12):1716-21.

Effects of methyl-, phenyl-, ethylmercury and mercurychlorid on immune cells of harbor seals (Phoca vitulina).

Kakuschke A, Valentine-Thon E, Fonfara S, Kramer K, Prange A.

GKSS Research Center Institute for Coastal Research, Max-Planck-Strasse 1, 21502 Geesthacht, Germany. antjekakuschke@web.de

Abstract

Mercury (Hg) is present in the marine environment as a natural metal often enhanced through human activities. Depending on its chemical form, Hg can cause a wide range of immunotoxic effects. In this study, the influence of methyl-, ethyl- and phenylmercury as well as mercurychloride on immune functions was evaluated. Two parameters of cellular immunity, proliferation and mRNA cytokine expression of interleukin-2, -4, and transforming growth factor beta, were investigated in harbor seal lymphocytes after in vitro exposure to Hg compounds. While all Hg compounds had a suppressive effect on proliferation, differences between juvenile and adult seals were found. Lymphocytes from juveniles showed a higher susceptibility to the toxic effect compared to lymphocytes from adults. Furthermore, the degree of inhibition of proliferation varied among the four Hg compounds. The organic compounds seem to be more immunotoxic than the inorganic compound. Finally, for the cytokine expression of methylmercury-incubated lymphocytes, time-dependent changes were observed, but no dose-dependency was found. Marine mammals of the North Sea are burdened with Hg, and lymphocytes of harbor seals may be functionally impaired by this metal. The present in vitro study provides baseline information for future studies on the immunotoxic effects of Hg on cellular immunity of harbor seals.

PMID: 20131603 [PubMed - indexed for MEDLINE]

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Cases J. 2009 Nov 18;2:199.

The usefulness of chelation therapy for the remission of symptoms caused by previous treatment with mercury-containing pharmaceuticals: a case report.

Corsello S, Fulgenzi A, Vietti D, Ferrero ME.

Dipartimento di Morfologia umana e Scienze Biomediche-Città Studi, Università di Milano, Via Mangiagalli, 31 20133, Milan, Italy. scorsello3@aol.com

Abstract

INTRODUCTION: A great deal of data regarding the toxicology of mercury has been recently reported. Although the most common human exposures to mercury are currently mercury vapour from amalgam tooth fillings, methylmercury from seafood and ethylmercury as a preservative in vaccines, in the past mercury compounds have been used in the treatment of syphilis.

CASE PRESENTATION: Mercury intoxication was found in a 67 year-old Italian man affected by neurological symptoms of apparently unknown origin. The patient developed syphilis forty years ago and then underwent therapy with mercurials to treat his chronic bacterial infection. We treated the patient with disodium edetate chelation therapy. Six months after the beginning of the therapy, the patient's neurological symptoms began to decrease, and were completely cured after two years of therapy.

CONCLUSION: This case supports the use of chelation therapy with disodium edetate to remove damages caused by mercury intoxication.

Don’t know what kind of mercury, however. It may not have been ethylmercury.

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FASEB J. 2009 Aug;23(8):2374-83. Epub 2009 Mar 23.

Cellular and mitochondrial glutathione redox imbalance in lymphoblastoid cells derived from children with autism.

James SJ, Rose S, Melnyk S, Jernigan S, Blossom S, Pavliv O, Gaylor DW.

Department of Pediatrics, University of Arkansas for Medical Sciences, Arkansas Children's Hospital Research Institute, 1120 Marshall St., Little Rock, AR 72202, USA. jamesjill@uams.edu

Abstract

Research into the metabolic phenotype of autism has been relatively unexplored despite the fact that metabolic abnormalities have been implicated in the pathophysiology of several other neurobehavioral disorders. Plasma biomarkers of oxidative stress have been reported in autistic children; however, intracellular redox status has not yet been evaluated. Lymphoblastoid cells (LCLs) derived from autistic children and unaffected controls were used to assess relative concentrations of reduced glutathione (GSH) and oxidized disulfide glutathione (GSSG) in cell extracts and isolated mitochondria as a measure of intracellular redox capacity. The results indicated that the GSH/GSSG redox ratio was decreased and percentage oxidized glutathione increased in both cytosol and mitochondria in the autism LCLs. Exposure to oxidative stress via the sulfhydryl reagent thimerosal resulted in a greater decrease in the GSH/GSSG ratio and increase in free radical generation in autism compared to control cells. Acute exposure to physiological levels of nitric oxide decreased mitochondrial membrane potential to a greater extent in the autism LCLs, although GSH/GSSG and ATP concentrations were similarly decreased in both cell lines. These results suggest that the autism LCLs exhibit a reduced glutathione reserve capacity in both cytosol and mitochondria that may compromise antioxidant defense and detoxification capacity under prooxidant conditions.

PMID: 19307255 [PubMed - indexed for MEDLINE]PMCID: PMC2717775Free PMC Article

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Biometals. 2009 Oct;22(5):697-700. Epub 2009 Feb 11.


Are toxic biometals destroying your children's future?

Drum DA.

poohpadon@wowway.com

Abstract

Cadmium, arsenic, lead, and mercury have been linked to autism, attention deficit disorder, mental retardation and death of children. Mercury in thimerosal found in many vaccines and flu shots contributes significantly to these problems. Decomposition of the thimerosal can produce more toxic compounds, either methylethylmercury or diethylmercury, in the body. These compounds have a toxicity level similar to dimethylmercury. Within the human body, a mitochondrial disorder may release the more toxic form of mercury internally. Young children and pregnant women must minimize internal exposure to the vaccines and flu shots containing mercury.

PMID: 19205900 [PubMed - indexed for MEDLINE]

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Exp Toxicol Pathol. 2009 Mar;61(2):133-6. Epub 2008 Sep 3.

Gender-selective toxicity of thimerosal.

Branch DR.

Departments of Medicine and Laboratory Medicine and Pathobiology, University of Toronto, Ontario, Canada. don.branch@utoronto.ca

Abstract

A recent report shows a correlation of the historical use of thimerosal in therapeutic immunizations with the subsequent development of autism; however, this association remains controversial. Autism occurs approximately four times more frequently in males compared to females; thus, studies of thimerosal toxicity should take into consideration gender-selective effects. The present study was originally undertaken to determine the maximum tolerated dose (MTD) of thimersosal in male and female CD1 mice. However, during the limited MTD studies, it became apparent that thimerosal has a differential MTD that depends on whether the mouse is male or female. At doses of 38.4-76.8mg/kg using 10% DMSO as diluent, seven of seven male mice compared to zero of seven female mice tested succumbed to thimerosal. Although the thimerosal levels used were very high, as we were originally only trying to determine MTD, it was completely unexpected to observe a difference of the MTD between male and female mice. Thus, our studies, although not directly addressing the controversy surrounding thimerosal and autism, and still preliminary due to small numbers of mice examined, provide, nevertheless, the first report of gender-selective toxicity of thimerosal and indicate that any future studies of thimerosal toxicity should take into consideration gender-specific differences.

PMID: 18771903 [PubMed - indexed for MEDLINE]

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Toxicol In Vitro. 2009 Sep;23(6):1092-9. Epub 2009 Jun 2.

Increase in intracellular Zn2+ concentration by thimerosal in rat thymocytes: intracellular Zn2+ release induced by oxidative stress.

Hashimoto E, Oyama TB, Oyama K, Nishimura Y, Oyama TM, Ueha-Ishibashi T, Okano Y, Oyama Y.

Laboratory of Cellular Signaling, Faculty of Integrated Arts and Sciences, The University of Tokushima, Tokushima 770-8502, Japan.

Abstract

Thimerosal (TMR), an ethylmercury-containing preservative in pharmaceutical products, was recently reported to increase intracellular Zn(2+) concentration. Therefore, some health concerns about the toxicity of TMR remain because of physiological and pathological roles of Zn(2+). To reveal the property of TMR-induced increase in intracellular Zn(2+) concentration, the effect of TMR on FluoZin-3 fluorescence, an indicator of intracellular Zn(2+), of rat thymocytes was examined. TMR at concentrations ranging from 0.3 microM to 10 microM increased the intensity of FluoZin-3 fluorescence in a concentration-dependent manner under external Ca(2+)- and Zn(2+)-free condition. The threshold concentration was 0.3-1 microM. The increase in the intensity was significant when TMR concentration was 1 microM or more. N,N,N',N'-Tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), a chelator for intracellular Zn(2+), completely attenuated the TMR-induced augmentation of FluoZin-3 fluorescence. Hydrogen peroxide (H(2)O(2)) and N-ethylmaleimide, reducing cellular thiol content, significantly increased FluoZin-3 fluorescence intensity and decreased 5-chloromethylfluorescein (5-CMF) fluorescence intensity, an indicator for cellular thiol. The correlation coefficient between TMR-induced augmentation of FluoZin-3 fluorescence and attenuation of 5-CMF fluorescence was -0.882. TMR also attenuated the 5-CMF fluorescence in the presence of TPEN. Simultaneous application of H(2)O(2) and TMR synergistically augmented the FluoZin-3 fluorescence. It is suggested that TMR increases intracellular Zn(2+) concentration via decreasing cellular thiol content.

PMID: 19497362 [PubMed - indexed for MEDLINE]

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J Ocul Pharmacol Ther. 2009 Apr;25(2):113-9.

Comparative toxicity of preservatives on immortalized corneal and conjunctival epithelial cells.

Epstein SP, Ahdoot M, Marcus E, Asbell PA.

Department of Ophthalmology, Mount Sinai Medical Center, New York, NY 10029-6574, USA. seth.epstein@mssm.edu

Abstract

PURPOSE: Nearly all eye drops contain preservatives to decrease contamination. Nonpreservatives such as disodium-ethylene diamine tetra-acetate (EDTA) and phosphate-buffered saline are also regularly added as buffering agents. These components can add to the toxicity of eye drops and cause ocular surface disease. To evaluate the potential toxicity of these common components and their comparative effects on the ocular surface, a tissue culture model utilizing immortalized corneal and conjunctival epithelial cells was utilized.

METHODS: Immortalized human conjunctival and corneal epithelial cells were grown. At confluency, medium was replaced with 100 microL of varying concentrations of preservatives: benzalkonium chloride (BAK), methyl paraben (MP), sodium perborate (SP), chlorobutanol (Cbl), and stabilized thimerosal (Thi); varying concentrations of buffer: EDTA; media (viable control); and formalin (dead control). After 1 h, solutions were replaced with 150 microL of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazonium bromide). After 4 h, solutions decanted, 100 microL of acid isopropanol added, and the optical density determined at 572 nm to evaluate cell viability.

RESULTS: Conjunctival and corneal cell toxicity was seen with all preservatives. Depending upon concentration, BAK exhibited from 56% to 89% toxicity. In comparison, Cbl exhibited from 50% to 86%, MP from 30% to 76%, SP from 23% to 59%, and Thi from 70% to 95%. EDTA with minimal toxicity (from 6% to 59%) was indistinguishable from SP.

CONCLUSIONS: Generally, the order of decreasing toxicity at the most commonly used concentrations: Thi (0.0025%) > BAK (0.025%) > Cbl (0.25%) > MP (0.01%) > SP (0.0025%) approximately EDTA (0.01%). Even at low concentration, these agents will cause some degree of ocular tissue damage.

PMID: 19284328 [PubMed - indexed for MEDLINE]PMCID: PMC2958436Free PMC Article

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J Med Food. 2009 Oct;12(5):1098-104.

Effects of glucan on immunosuppressive actions of mercury.

Vetvicka V, Vetvickova J.

Department of Pathology, University of Louisville, Louisville, Kentucky 40202, USA. vaclav@iglou.com

Abstract

Global cycling of mercury results in the presence of mercury salts in the environment. The well-established negative effects of mercury on the immune system led us to the study whether natural immunomodulator glucan can overcome the immunosuppressive effects of mercury. Two types of mercury, thimerosal and mercury acetate, were administered in a dose of 2-8 mg/L of drinking water to mice. After 2 weeks, all mice exhibited profound suppression of both cellular (phagocytosis, natural killer cell activity, mitogen-induced proliferation, and expression of CD markers) and humoral (antibody formation and secretion of interleukin-6, interleukin-12, and interferon-gamma) responses. The mice were then fed with a diet containing a standard dose of glucan. Our results showed that simultaneous treatment with mercury and glucan resulted in significantly lower immunotoxic effects of mercury, which suggests that glucans can be successfully used as a natural remedy of low-level exposure to mercury.

PMID: 19857075 [PubMed - indexed for MEDLINE]

Wonder what, if any, cell damage occurs when injected.

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Curr Med Chem. 2008;15(28):3000-10.

Kawasaki's disease, acrodynia, and mercury.

Mutter J, Yeter D.

Department of Environmental and Complementary Medicine, Salusmed Medical Center, Wieslistrasse 34, CH - 8267 Berlingen, Switzerland. jo.mutter@web.de

Abstract

A superantigen or autoimmunity has been hypothesized to be the main cause of the Kawasaki's Disease but the etiology is unknown. Medical literature, epidemiological findings, and some case reports have suggested that mercury may play a pathogenic role. Several patients with Kawasaki's Disease have presented with elevated urine mercury levels compared to matched controls. Most symptoms and diagnostic criteria which are seen in children with acrodynia, known to be caused by mercury, are similar to those seen in Kawasaki's Disease. Genetic depletion of glutathione S-transferase , a susceptibility marker for Kawasaki's Disease, is known to be also a risk factor for acrodynia and may also increase susceptibility to mercury . Coinciding with the largest increase (1985-1990) of thimerosal (49.6% ethyl mercury) in vaccines, routinely given to infants in the U.S. by 6 months of age (from 75microg to 187.5microg), the rates of Kawasaki's Disease increased ten times, and, later (1985-1997), by 20 times. Since 1990 88 cases of patients developing Kawasaki's Disease some days after vaccination have been reported to the Centers of Disease Control (CDC) including 19% manifesting symptoms the same day. The presented pathogenetic model may lead to new preventive- and therapeutic strategies for Kawasaki's disease.

PMID: 19075648 [PubMed - indexed for MEDLINE]

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Toxicol In Vitro. 2008 Jun;22(4):927-34. Epub 2008 Feb 1.

Genotoxicity of thimerosal in cultured human lymphocytes with and without metabolic activation sister chromatid exchange analysis proliferation index and mitotic index.

Eke D, Celik A.

Mersin University, Faculty of Science and Letters, Department of Biology, 33343 Mersin, Turkey.

Abstract

Thimerosal is an antiseptic containing 49.5% of ethyl mercury that has been used for years as a preservative in many infant vaccines and in flu vaccines. Thimerosal is an organic mercurial compound used as a preservative in biomedical preparations. In this study, we evaluated the genotoxic effect of thimerosal in cultured human peripheral blood lymphocytes using sister chromatid exchange analysis in culture conditions with and without S9 metabolic activation. This study is the first report investigating the genotoxic effects of thimerosal in cultured human peripheral blood lymphocyte cells using sister chromatid exchange analysis. An analysis of variance test (ANOVA) was performed to evaluate the results. Significant induction of sister chromatid exchanges was seen at concentrations between 0.2 and 0.6 microg/ml of thimerosal compared with negative control. A significant decrease (p<0.001) in mitotic index (MI) and proliferation index (PRI) as well as an increase in SCE frequency (p<0.001) was observed compared with control cultures. Our results indicate the genotoxic and cytotoxic effect of TH in cultured human peripheral blood lymphocytes at tested doses in cultures with/without S9 fraction.

PMID: 18321677 [PubMed - indexed for MEDLINE]

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Chem Res Toxicol. 2008 Feb;21(2):483-93. Epub 2008 Jan 16.

Thiol-modulated mechanisms of the cytotoxicity of thimerosal and inhibition of DNA topoisomerase II alpha.

Wu X, Liang H, O'Hara KA, Yalowich JC, Hasinoff BB.

Faculty of Pharmacy, University of Manitoba, 50 Sifton Road, Winnipeg, Manitoba, R3T 2N2, Canada.

Abstract

Thimerosal is an organic mercury compound that is widely used as a preservative in vaccines and other solution formulations. The use of thimerosal has caused concern about its ability to cause neurological abnormalities due to mercury accumulation during a normal schedule of childhood vaccinations. While the chemistry and the biological effects of methylmercury have been well-studied, those of thimerosal have not. Thimerosal reacted rapidly with cysteine, GSH, human serum albumin, and single-stranded DNA to form ethylmercury adducts that were detectable by mass spectrometry. These results indicated that thimerosal would be quickly metabolized in vivo because of its reactions with protein and nonprotein thiols. Thimerosal also potently inhibited the decatenation activity of DNA topoisomerase II alpha, likely through reaction with critical free cysteine thiol groups. Thimerosal, however, did not act as a topoisomerase II poison and the lack of cross-resistance with a K562 cell line with a decreased level of topoisomerase II alpha (K/VP.5 cells) suggested that inhibition of topoisomerase II alpha was not a significant mechanism for the inhibition of cell growth. Depletion of intracellular GSH with buthionine sulfoximine treatment greatly increased the K562 cell growth inhibitory effects of thimerosal, which showed that intracellular glutathione had a major role in protecting cells from thimerosal. Pretreatment of thimerosal with glutathione did not, however, change its K562 cell growth inhibitory effects, a result consistent with the rapid exchange of the ethylmercury adduct among various thiol-containing cellular reactants. Thimerosal-induced single and double strand breaks in K562 cells were consistent with a rapid induction of apoptosis. In conclusion, these studies have elucidated some of the chemistry and biological activities of the interaction of thimerosal with topoisomerase II alpha and protein and nonprotein thiols and with DNA.

PMID: 18197631 [PubMed - indexed for MEDLINE]

Apoptosis is “programmed cell death”.

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Altern Ther Health Med. 2008 Nov-Dec;14(6):46-53.

A possible central mechanism in autism spectrum disorders, part 1.

Blaylock RL.

Belhaven College, Jackson, Mississippi, USA.

Abstract

The autism spectrum disorders (ASD) are a group of related neurodevelopmental disorders that have been increasing in incidence since the 1980s. Despite a considerable amount of data being collected from cases, a central mechanism has not been offered. A careful review of ASD cases discloses a number of events that adhere to an immunoexcitotoxic mechanism. This mechanism explains the link between excessive vaccination, use of aluminum and ethylmercury as vaccine adjuvants, food allergies, gut dysbiosis, and abnormal formation of the developing brain. It has now been shown that chronic microglial activation is present in autistic brains from age 5 years to age 44 years. A considerable amount of evidence, both experimental and clinical, indicates that repeated microglial activation can initiate priming of the microglia and that subsequent stimulation can produce an exaggerated microglial response that can be prolonged. It is also known that one phenotypic form of microglia activation can result in an outpouring of neurotoxic levels of the excitotoxins, glutamate and quinolinic acid. Studies have shown that careful control of brain glutamate levels is essential to brain pathway development and that excesses can result in arrest of neural migration, as well as dendritic and synaptic loss. It has also been shown that certain cytokines, such as TNF-alpha, can, via its receptor, interact with glutamate receptors to enhance the neurotoxic reaction. To describe this interaction I have coined the term immunoexcitotoxicity, which is described in this article.

PMID: 19043938 [PubMed - indexed for MEDLINE]

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J Neurol Sci. 2008 Aug 15;271(1-2):110-8. Epub 2008 May 15.

Thimerosal exposure in infants and neurodevelopmental disorders: an assessment of computerized medical records in the Vaccine Safety Datalink.

Young HA, Geier DA, Geier MR.

The George Washington University School of Public Health and Health Services, Department of Epidemiology and Biostatistics, United States.

Abstract

The study evaluated possible associations between neurodevelopmental disorders (NDs) and exposure to mercury (Hg) from Thimerosal-containing vaccines (TCVs) by examining the automated Vaccine Safety Datalink (VSD). A total of 278,624 subjects were identified in birth cohorts from 1990-1996 that had received their first oral polio vaccination by 3 months of age in the VSD. The birth cohort prevalence rate of medically diagnosed International Classification of Disease, 9th revision (ICD-9) specific NDs and control outcomes were calculated. Exposures to Hg from TCVs were calculated by birth cohort for specific exposure windows from birth-7 months and birth-13 months of age. Poisson regression analysis was used to model the association between the prevalence of outcomes and Hg doses from TCVs. Consistent significantly increased rate ratios were observed for autism, autism spectrum disorders, tics, attention deficit disorder, and emotional disturbances with Hg exposure from TCVs. By contrast, none of the control outcomes had significantly increased rate ratios with Hg exposure from TCVs. Routine childhood vaccination should be continued to help reduce the morbidity and mortality associated with infectious diseases, but efforts should be undertaken to remove Hg from vaccines. Additional studies should be conducted to further evaluate the relationship between Hg exposure and NDs.

PMID: 18482737 [PubMed - indexed for MEDLINE]

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Neurotoxicology. 2008 May;29(3):532-45. Epub 2008 Feb 23.

Immunologic and neurodevelopmental susceptibilities of autism.

Pessah IN, Seegal RF, Lein PJ, LaSalle J, Yee BK, Van De Water J, Berman RF.

School of Veterinary Medicine, University of California, Davis, CA 95616, USA. inpessah@ucdavis.edu

Abstract

Symposium 5 focused on research approaches that are aimed at understanding common patterns of immunological and neurological dysfunction contributing to neurodevelopmental disorders such as autism and ADHD. The session focused on genetic, epigenetic, and environmental factors that might act in concert to influence autism risk, severity and co-morbidities, and immunological and neurobiological targets as etiologic contributors. The immune system of children at risk of autism may be therefore especially susceptible to psychological stressors, exposure to chemical triggers, and infectious agents. Identifying early biomarkers of risk provides tangible approaches toward designing studies in animals and humans that yield a better understanding of environmental risk factors, and can help identify rational intervention strategies to mitigate these risks.

PMID: 18394707 [PubMed - indexed for MEDLINE]PMCID: PMC2475601Free PMC Article http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2475601/?tool=pubmed

See Section VII – “Examination of Thimerosal Effects in neonatal SJL/J Mice at Vaccination-Associated Exposure Levels”
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J Toxicol Environ Health A. 2007 Dec;70(24):2092-5.

Evaluation of cytotoxicity attributed to thimerosal on murine and human kidney cells.

Park EK, Mak SK, Kültz D, Hammock BD.

Department of Entomology and Cancer Research Center, University of California, Davis, California, USA. eunkee.park@ddb.nsw.gov.au

Abstract

Renal inner medullary collecting duct cells (mIMCD3) and human embryonic kidney cells (HEK293) were used for cytoscreening of thimerosal and mercury chloride (HgCl2). Thimerosal and HgCl2 acted in a concentration-dependent manner. In mIMCD3 cells the 24-h LC50 values for thimerosal, thiosalicylic acid, 2,2-dithiosalicylic acid, and 2-sulfobenzoic acid were 2.9, 2200, >1000, and >10,000 microM, respectively. The 24-h LC50 value for HgCl2 in mIMCD3 cells was 40 microM. In HEK293 cells, the 24-h LC50 value for thimerosal was 9.5 microM. These data demonstrate that the higher cytotoxicity produced by thimerosal on renal cells with respect to similar compounds without Hg may be related to this metal content. The present study also establishes mIMCD3 cells as a valuable model for evaluation of cytotoxicity of nephrotoxic compounds.

PMID: 18049999 [PubMed - indexed for MEDLINE]

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Toxicol Sci. 2007 Nov;100(1):109-17. Epub 2007 Aug 13.

Thimerosal-induced apoptosis in human SCM1 gastric cancer cells: activation of p38 MAP kinase and caspase-3 pathways without involvement of [Ca2+]i elevation.

Liu SI, Huang CC, Huang CJ, Wang BW, Chang PM, Fang YC, Chen WC, Wang JL, Lu YC, Chu ST, Chou CT, Jan CR.

Department of Surgery, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan 813.

Abstract

Thimerosal is a mercury-containing preservative in some vaccines. The effect of thimerosal on human gastric cancer cells is unknown. This study shows that in cultured human gastric cancer cells (SCM1), thimerosal reduced cell viability in a concentration- and time-dependent manner. Thimerosal caused apoptosis as assessed by propidium iodide-stained cells and caspase-3 activation. Although immunoblotting data revealed that thimerosal could activate the phosphorylation of extracellular signal-regulated kinase, c-Jun NH2-terminal protein kinase, and p38 mitogen-activated protein kinase (p38 MAPK), only SB203580 (a p38 MAPK inhibitor) partially prevented cells from apoptosis. Thimerosal also induced [Ca2+](i) increases via Ca2+ influx from the extracellular space. However, pretreatment with (bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetate)/AM, a Ca2+ chelator, to prevent thimerosal-induced [Ca2+](i) increases did not protect cells from death. The results suggest that in SCM1 cells, thimerosal caused Ca2+-independent apoptosis via phosphorylating p38 MAPK resulting in caspase-3 activation.

PMID: 17698513 [PubMed - indexed for MEDLINE]Free Article

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Toxicology. 2007 Feb 28;231(1):40-57. Epub 2006 Nov 25.

Cell death and cytotoxic effects in YAC-1 lymphoma cells following exposure to various forms of mercury.

Yole M, Wickstrom M, Blakley B.

Department of Veterinary Biomedical Sciences, Western College of Veterinary Medicine, 52 Campus Drive, University of Saskatchewan, Saskatoon SK S7N 5B4, Canada. yole@sask.usask.ca

Erratum in:

  • Toxicology. 2008 Jan 14;243(1-2):244-5.

Abstract

The effects of 1 min-4 h exposures to four Hg compounds (mercuric chloride [HgCl2], methyl mercuric chloride [CH3HgCl], p-chloromercuribenzoate [p-CMB] and thimerosal [TMS; ethylmercurithiosalicylate]) on cell death, microtubules, actin, CD3 receptor expression, protein tyrosine phosphorylation (PTyr-P) and intracellular calcium ([Ca2+]i) levels were investigated in YAC-1 lymphoma cells using flow cytometry. YOPRO-1 (YP) and propidium iodide (PI) dye uptake indicated all forms of Hg tested were toxic at concentrations ranging from 25.8-48.4 microM, with two distinct patterns of effects. Early apoptosis was prolonged for CH3HgCl- and TMS-treated cells, with more than 50% remaining YP+/PI- after 4h. Both CH3HgCl and TMS induced complete loss of beta-tubulin fluorescence, indicative of microtubule depolymerization and inhibition of tubulin synthesis and/or beta-tubulin degradation, while F-actin fluorescence diminished to a lesser degree and only after loss beta-tubulin. CH3HgCl and TMS induced an almost immediate two-fold increase in CD3 fluorescence, with levels returning to baseline within minutes. With continued exposure, CD3 fluorescence was reduced to approximately 50% of baseline values. Both compounds also increased PTyr-P two- to three-fold immediately, with levels returning to baseline at 4h. Similarly, two- to three-fold increases in [Ca2+]i were noted after 1 min exposure. [Ca2+]i increased progressively, reaching levels five- to eight-fold greater than control values. In contrast, dye uptake was delayed with HgCl2 and p-CMB, although cell death proceeded rapidly, with almost all non-viable cells being late apoptotic (YP+/PI+) by 4h. p-CMB produced early reductions in F-actin, and after 4h, complete loss of F-actin with only partial reduction of total beta-tubulin was seen with both p-CMB and HgCl2. HgCl2 reduced CD3 expression and PTyr-P slightly within minutes, while p-CMB produced similar effects on CD3 only at 4h, at which time PTyr-P was increased two- to three-fold. Both compounds increased [Ca2+]i within minutes, though levels remained under twice the baseline concentration after 15 min exposure. With continued exposure, [Ca2+]i increased to levels two- to five-fold greater than control values. These findings indicate the two groups of Hg compounds may induce cell death by distinct pathways, reflecting interactions with different cellular targets leading to cell death.

PMID: 17210217 [PubMed - indexed for MEDLINE]

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Toxicology. 2007 Jan 5;229(1-2):23-32. Epub 2006 Sep 24.

Dose and Hg species determine the T-helper cell activation in murine autoimmunity.

Havarinasab S, Björn E, Ekstrand J, Hultman P.

Molecular and Immunological Pathology (AIR), Department of Molecular and Clinical Medicine, Linköping University, SE-581 85 Linköping, Sweden. saiha@imk.liu.se

Abstract

Inorganic mercury (mercuric chloride--HgCl(2)) induces in mice an autoimmune syndrome (HgIA) with T cell-dependent polyclonal B cell activation and hypergammaglobulinemia, dose- and H-2-dependent production of autoantibodies targeting the 34 kDa nucleolar protein fibrillarin (AFA), and systemic immune-complex deposits. The organic mercury species methylmercury (MeHg) and ethylmercury (EtHg--in the form of thimerosal) induce AFA, while the other manifestations of HgIA seen after treatment with HgCl(2) are present to varying extent. Since these organic Hg species are converted to the autoimmunogen Hg(2+) in the body, their primary autoimmunogen potential is uncertain and the subject of this study. A moderate dose of HgCl(2) (8 mg/L drinking water--internal dose 148 micro gHg/kg body weight [bw]/day) caused the fastest AFA response, while the induction was delayed after higher (25 mg/L) and lower (1.5 and 3 mg/L) doses. The lowest dose of HgCl(2) inducing AFA was 1.5 mg/L drinking water which corresponded to a renal Hg(2+) concentration of 0.53 micro g/g. Using a dose of 8 mg HgCl(2)/L this threshold concentration was reached within 24 h, and a consistent AFA response developed after 8-10 days. The time lag for the immunological part of the reaction leading to a consistent AFA response was therefore 7-9 days. A dose of thimerosal close to the threshold dose for induction of AFA (2 mg/L drinking water--internal dose 118 micro gHg/kg bw per day), caused a renal Hg(2+) concentration of 1.8 micro g/g. The autoimmunogen effect of EtHg might therefore be entirely due to Hg(2+) formed from EtHg in the body. The effect of organic and inorganic Hg species on T-helper type 1 and type 2 cells during induction of AFA was assessed as the presence and titre of AFA of the IgG1 and IgG2a isotype, respectively. EtHg induced a persistent Th1-skewed response irrespectively of the dose and time used. A low daily dose of HgCl(2) (1.5-3 mg/L) caused a Th1-skewed AFA response, while a moderate dose (8 mg/L) after 2 weeks resulted in a balanced or even Th2-skewed response. Higher daily doses of HgCl(2) (25 mg/L) caused a balanced Th2-Th1 response already from onset. In conclusion, while metabolically formed Hg(2+) might be the main AFA-inducing factor also after treatment with EtHg, the quality of the Hg-induced AFA response is modified by the species of Hg as well as the dose.

PMID: 17084957 [PubMed - indexed for MEDLINE]

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J Matern Fetal Neonatal Med. 2007 May;20(5):385-90.

A prospective study of thimerosal-containing Rho(D)-immune globulin administration as a risk factor for autistic disorders.

Geier DA, Geier MR.

The Institute of Chronic Illnesses, Silver Spring, MD, USA.

Abstract

BACKGROUND: This study evaluated the relationship between prenatal mercury exposure from thimerosal (49.55% mercury by weight)-containing Rho(D)-immune globulins (TCRs) and autism spectrum disorders (ASDs).

METHODS: The Institutional Review Board of the Institute for Chronic Illnesses approved the present study. A total of 53 consecutive non-Jewish Caucasian patients with ASDs (Diagnostic and statistical manual of mental disorders, fourth ed. - DSM IV) born between 1987 and 2001 who presented to the Genetic Centers of America for outpatient genetic/developmental evaluations were prospectively collected from June 1, 2005 through March 31, 2006. Imaging and laboratory testing were conducted on each patient to rule out other causal factors for their ASDs. As race-matched controls, the frequency of Rh negativity was determined from 926 non-Jewish Caucasian pregnant women who had presented for outpatient prenatal genetics care to the Genetic Centers of America between 1980 and 1989.

RESULTS: Children with ASDs (28.30%) were significantly more likely (odds ratio 2.35, 95% confidence interval 1.17-4.52, p < 0.01) to have Rh-negative mothers than controls (14.36%). Each ASD patient's mother was determined to have been administered a TCR during her pregnancy.

CONCLUSION: The results provide insights into the potential role prenatal mercury exposure may play in some children with ASDs.

PMID: 17674242 [PubMed - indexed for MEDLINE]

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J Toxicol Environ Health A. 2007 May 15;70(10):837-51.

A case series of children with apparent mercury toxic encephalopathies manifesting with clinical symptoms of regressive autistic disorders.

Geier DA, Geier MR.

Institute of Chronic Illnesses, Inc., Silver Spring, Maryland, USA.

Abstract

Impairments in social relatedness and communication, repetitive behaviors, and stereotypic abnormal movement patterns characterize autism spectrum disorders (ASDs). It is clear that while genetic factors are important to the pathogenesis of ASDs, mercury exposure can induce immune, sensory, neurological, motor, and behavioral dysfunctions similar to traits defining or associated with ASDs. The Institutional Review Board of the Institute for Chronic Illnesses (Office for Human Research Protections, U.S. Department of Health and Human Services, IRB number IRB00005375) approved the present study. A case series of nine patients who presented to the Genetic Centers of America for a genetic/developmental evaluation are discussed. Eight of nine patients (one patient was found to have an ASD due to Rett's syndrome) (a) had regressive ASDs; (b) had elevated levels of androgens; (c) excreted significant amounts of mercury post chelation challenge; (d) had biochemical evidence of decreased function in their glutathione pathways; (e) had no known significant mercury exposure except from Thimerosal-containing vaccines/Rho(D)-immune globulin preparations; and (f) had alternate causes for their regressive ASDs ruled out. There was a significant dose-response relationship between the severity of the regressive ASDs observed and the total mercury dose children received from Thimerosal-containing vaccines/Rho (D)-immune globulin preparations. Based upon differential diagnoses, 8 of 9 patients examined were exposed to significant mercury from Thimerosal-containing biologic/vaccine preparations during their fetal/infant developmental periods, and subsequently, between 12 and 24 mo of age, these previously normally developing children suffered mercury toxic encephalopathies that manifested with clinical symptoms consistent with regressive ASDs. Evidence for mercury intoxication should be considered in the differential diagnosis as contributing to some regressive ASDs.

PMID: 17454560 [PubMed - indexed for MEDLINE]

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Toxicol Sci. 2006 Jul;92(1):246-53. Epub 2006 Apr 19.

Thimerosal induces apoptosis in a neuroblastoma model via the cJun N-terminal kinase pathway.

Herdman ML, Marcelo A, Huang Y, Niles RM, Dhar S, Kiningham KK.

Department of Pharmacology, Joan C. Edwards School of Medicine, Marshall University, 1542 Spring Valley Drive, Huntington, WV 25704, USA.

Abstract

The cJun N-terminal kinase (JNK)-signaling pathway is activated in response to a variety of stimuli, including environmental insults, and has been implicated in neuronal apoptosis. In this study, we investigated the role that the JNK pathway plays in neurotoxicity caused by thimerosal, an ethylmercury-containing preservative. SK-N-SH cells treated with thimerosal (0-10 microM) showed an increase in the phosphorylated (active) form of JNK and cJun with 5 and 10 microM thimerosal treatment at 2 and 4 h. To examine activator protein-1 (AP-1) transcription, cells were transfected with a pGL2 vector containing four AP-1 consensus sequences and then treated with thimerosal (0-2.5 microM) for 24 h. Luciferase studies showed an increase in AP-1 transcriptional activity upon thimerosal administration. To determine the components of the AP-1 complex, cells were transfected with a dominant negative to either cFos (A-Fos) or cJun (TAM67). Reporter analysis showed that TAM67, but not A-Fos, decreased AP-1 transcriptional activity, indicating a role for cJun in this pathway. To assess which components are essential to apoptosis, cells were treated with a cell-permeable JNK inhibitor II (SP600125) or transfected with TAM67, and the downstream effectors of apoptosis were analyzed. Cells pretreated with SP600125 showed decreases in activation of caspases 9 and 3, decreases in degradation of poly(ADP-ribose) polymerase (PARP), and decreased levels of proapoptotic Bim, in comparison to cells treated with thimerosal alone. However, cells transfected with TAM67 showed no changes in those same components. Taken together, these results indicate that thimerosal-induced neurotoxicity occurs through the JNK-signaling pathway, independent of cJun activation, leading ultimately to apoptotic cell death.

PMID: 16624850 [PubMed - indexed for MEDLINE]Free Article
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Mol Carcinog. 2006 Sep;45(9):657-66.

Thimerosal induces apoptosis and G2/M phase arrest in human leukemia cells.

Woo KJ, Lee TJ, Bae JH, Jang BC, Song DK, Cho JW, Suh SI, Park JW, Kwon TK.

Department of Immunology, School of Medicine, Keimyung University, Taegu, South Korea.

Abstract

Thimerosal is an organomercury compound with sulfhydryl-reactive properties. The ability of thimerosal to act as a sulfhydryl group is related to the presence of mercury. Due to its antibacterial effect, thimerosal is widely used as preservatives and has been reported to cause chemically mediated side effects. In the present study, we showed that the molecular mechanism of thimerosal induced apoptosis in U937 cells. Thimerosal was shown to be responsible for the inhibition of U937 cells growth by inducing apoptosis. Treatment with 2.5-5 microM thimerosal but not thiosalicylic acid (structural analog of thimerosal devoid of mercury) for 12 h produced apoptosis, G(2)/M phase arrest, and DNA fragmentation in a dose-dependent manner. Treatment with caspase inhibitor significantly reduced thimerosal-induced caspase 3 activation. In addition, thimerosal-induced apoptosis was attenuated by antioxidant Mn (III) meso-tetrakis (4-benzoic acid) porphyrin (Mn-TBAP). These data indicate that the cytotoxic effect of thimerosal on U937 cells is attributable to the induced apoptosis and that thimerosal-induced apoptosis is mediated by reactive oxygen species generation and caspase-3 activation.

PMID: 16649253 [PubMed - indexed for MEDLINE]
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Neurotoxicology. 2006 Sep;27(5):685-92. Epub 2006 Jun 16.

Cultured lymphocytes from autistic children and non-autistic siblings up-regulate heat shock protein RNA in response to thimerosal challenge.

Walker SJ, Segal J, Aschner M.

Department of Physiology and Pharmacology, Wake Forest University School of Medicine, Winston-Salem, NC 27156, USA. swalker@wfubmc.edu

Abstract

There are reports suggesting that some autistic children are unable to mount an adequate response following exposure to environmental toxins. This potential deficit, coupled with the similarity in clinical presentations of autism and some heavy metal toxicities, has led to the suggestion that heavy metal poisoning might play a role in the etiology of autism in uniquely susceptible individuals. Thimerosal, an anti-microbial preservative previously added routinely to childhood multi-dose vaccines, is composed of 49.6% ethyl mercury. Based on the levels of this toxin that children receive through routine immunization schedules in the first years of life, it has been postulated that thimerosal may be a potential triggering mechanism contributing to autism in susceptible individuals. One potential risk factor in these individuals may be an inability to adequately up-regulate metallothionein (MT) biosynthesis in response to presentation of a heavy metal challenge. To investigate this hypothesis, cultured lymphocytes (obtained from the Autism Genetic Resource Exchange, AGRE) from autistic children and non-autistic siblings were challenged with either 10 microM ethyl mercury, 150 microM zinc, or fresh media (control). Following the challenge, total RNA was extracted and used to query "whole genome" DNA microarrays. Cultured lymphocytes challenged with zinc responded with an impressive up-regulation of MT transcripts (at least nine different MTs were over-expressed) while cells challenged with thimerosal responded by up-regulating numerous heat shock protein transcripts, but not MTs. Although there were no apparent differences between autistic and non-autistic sibling responses in this very small sampling group, the differences in expression profiles between those cells treated with zinc versus thimerosal were dramatic. Determining cellular response, at the level of gene expression, has important implications for the understanding and treatment of conditions that result from exposure to neurotoxic compounds.

PMID: 16870260 [PubMed - indexed for MEDLINE]

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Altern Ther Health Med. 2006 Jul-Aug;12(4):16-7.

Are mercury amalgam fillings safe for children? An evaluation of recent research results.

Rode D.

EcoNugenics Inc, Santa Rosa, California, USA.

Abstract

Two recent clinical trials on the safety of amalgam fillings in children found no evidence of harmful effects from mercury-containing dental fillings after following children for 5-7 years. This review suggests the studies' results are limited by (1) sample sizes that were too small to allow detection of genetic variations in mercury toxicity at a rate of 1 in 100 or lower, (2) a lack of control for other sources of mercury, and (3) a population that may have been skewed by excluding children with autism during a time when autism was escalating due, in part, to increased frequency of thimerosal-containing vaccine use.

PMID: 16862738 [PubMed - indexed for MEDLINE]

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Neuro Endocrinol Lett. 2006 Aug;27(4):401-13.

A meta-analysis epidemiological assessment of neurodevelopmental disorders following vaccines administered from 1994 through 2000 in the United States.

Geier DA, Geier MR.

The Institute for Chronic Illnesses, Inc., Silver Spring, MD 20905, USA. mgeier@comcast.net

Abstract

BACKGROUND: Thimerosal is an ethylmercury-containing compound (49.6% mercury by weight) used as at the preservative level in vaccines (0.005% to 0.01%).

METHODS: Statistical modeling in a meta-analysis epidemiological assessment of the Vaccine Adverse Event Reporting System (VAERS) for neurodevelopment disorders (NDs) reported following Diphtheria-Tetanus-whole-cell-Pertussis (DTP) vaccines in comparison to Diphtheria-Tetanus-whole-cell-Pertussis-Haemophilus Influenzae Type b (DTPH) vaccines (administered: 1994-1997) and following Thimerosal-containing Diphtheria-Tetanus-acellular-Pertussis (DTaP), vaccines in comparison to Thimerosal-free DTaP vaccines (administered: 1997-2000), was undertaken.

RESULTS: Significantly increased adjusted (sex, age, vaccine type, vaccine manufacturer) risks of autism, speech disorders, mental retardation, personality disorders, thinking abnormalities, ataxia, and NDs in general, with minimal systematic error or confounding, were associated with TCV exposure.

CONCLUSION: It is clear from the results of the present epidemiological study and other recently published data associating mercury exposure with childhood NDs, additional ND research should be undertaken in the context of evaluating mercury-associated exposures, especially from Thimerosal-containing vaccines.

PMID: 16807526 [PubMed - indexed for MEDLINE]

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Acta Microbiol Immunol Hung. 2005;52(1):95-103.

Being on the track of thimerosal. Review.

Mádi A.

Signalling and Apoptosis Research Group of the Hungarian Academy of Sciences, University of Debrecen, Nagyerdei krt. 98, H-4012 Debrecen, Hungary. madi@indi.biochem.dote.hu

Abstract

The common preservative thimerosal is one of the most important organic mercury compounds human populations are exposed to. It has toxic effect on several cell lines, and it also induces programmed cell death in in vitro experiments. Association is suggested between application of thimerosal-containing vaccines and the occurrence of neurodevelopmental disorders, like autism. While specific recommendations were made to eliminate thimerosal from vaccines, consistent evidence is still lacking for an association of exposure and disease. Unfortunately, it is very hard to study the molecular background of complex human diseases directly; however, investigations on more simple model organisms may lead to a better understanding of thimerosal as a possible disease inducing factor.

PMID: 15957237 [PubMed - indexed for MEDLINE]

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Med Sci Monit. 2005 Apr;11(4):CR160-70. Epub 2005 Mar 24.

A two-phased population epidemiological study of the safety of thimerosal-containing vaccines: a follow-up analysis.

Geier DA, Geier MR.

MedCon, Inc., USA.

Abstract

BACKGROUND: Thimerosal is an ethylmercury-containing preservative in vaccines. Toxicokinetic studies have shown children received doses of mercury from thimerosal-containing vaccines (TCVs) that were in excess of safety guidelines. Previously, an ecological study showing a significant association between TCVs and neurodevelopmental disorders (NDs) in the US was published in this journal.

MATERIAL/METHODS: A two phased population-based epidemiological study was undertaken. Phase one evaluated reported NDs to the Vaccine Adverse Event Reporting System (VAERS) following thimerosal-containing Diphtheria-Tetanus-acellular-Pertussis (DTaP) vaccines in comparison to thimerosal-free DTaP vaccines administered from 1997 through 2001. Phase two evaluated the automated Vaccine Safety Datalink (VSD) for cumulative exposures to mercury from TCVs at 1-, 2-, 3-, and 6-months-of-age for infants born from 1992 through 1997 and the eventual risk of developing NDs.

RESULTS: Phase one showed significantly increased risks for autism, speech disorders, mental retardation, personality disorders, and thinking abnormalities reported to VAERS following thimerosal-containing DTaP vaccines in comparison to thimerosal-free DTaP vaccines. Phase two showed significant associations between cumulative exposures to thimerosal and the following types of NDs: unspecified developmental delay, tics, attention deficit disorder (ADD), language delay, speech delay, and neurodevelopmental delays in general.

CONCLUSIONS: This study showed that exposure to mercury from TCVs administered in the US was a consistent significant risk factor for the development of NDs. It is clear from these data and other recent publications linking TCVs with NDs that additional ND research should be undertaken in the context of evaluating mercury-associated exposures and thimerosal-free vaccines should be made available.

PMID: 15795695 [PubMed - indexed for MEDLINE]

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Neuro Endocrinol Lett. 2005 Oct;26(5):439-46.

Mercury and autism: accelerating evidence?

Mutter J, Naumann J, Schneider R, Walach H, Haley B.

Institute for Environmental Medicine and Hospital Epidemiology, University Hospital Freiburg, Germany. joachim.mutter@uniklinik-freiburg.de

Abstract

The causes of autism and neurodevelopmental disorders are unknown. Genetic and environmental risk factors seem to be involved. Because of an observed increase in autism in the last decades, which parallels cumulative mercury exposure, it was proposed that autism may be in part caused by mercury. We review the evidence for this proposal. Several epidemiological studies failed to find a correlation between mercury exposure through thimerosal, a preservative used in vaccines, and the risk of autism. Recently, it was found that autistic children had a higher mercury exposure during pregnancy due to maternal dental amalgam and thimerosal-containing immunoglobulin shots. It was hypothesized that children with autism have a decreased detoxification capacity due to genetic polymorphism. In vitro, mercury and thimerosal in levels found several days after vaccination inhibit methionine synthetase (MS) by 50%. Normal function of MS is crucial in biochemical steps necessary for brain development, attention and production of glutathione, an important antioxidative and detoxifying agent. Repetitive doses of thimerosal leads to neurobehavioral deteriorations in autoimmune susceptible mice, increased oxidative stress and decreased intracellular levels of glutathione in vitro. Subsequently, autistic children have significantly decreased level of reduced glutathione. Promising treatments of autism involve detoxification of mercury, and supplementation of deficient metabolites.

PMID: 16264412 [PubMed - indexed for MEDLINE]

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Environ Health Perspect. 2005 Aug;113(8):1015-21.

Comparison of blood and brain mercury levels in infant monkeys exposed to methylmercury or vaccines containing thimerosal.

Burbacher TM, Shen DD, Liberato N, Grant KS, Cernichiari E, Clarkson T.

Department of Environmental and Occupational Health Sciences, School of Public Health and Community Medicine, University of Washington, Seattle, Washington 98195, USA. tmb@u.washington.edu

Abstract

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.

PMID: 16079072 [PubMed - indexed for MEDLINE]PMCID: PMC1280342Free PMC Article

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Neurotoxicology. 2005 Jun;26(3):407-16.

Mitochondrial mediated thimerosal-induced apoptosis in a human neuroblastoma cell line (SK-N-SH).

Humphrey ML, Cole MP, Pendergrass JC, Kiningham KK.

Department of Pharmacology, Joan C. Edwards School of Medicine, Marshall University, Huntington, WV 25704-9388, USA.

Abstract

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.

PMID: 15869795 [PubMed - indexed for MEDLINE]

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Toxicol Sci. 2005 Jul;86(1):132-40. Epub 2005 Apr 20.

Effects of thimerosal on NGF signal transduction and cell death in neuroblastoma cells.

Parran DK, Barker A, Ehrich M.

Virginia-Maryland Regional College of Veterinary Medicine, Laboratory for Neurotoxicity Studies, Virginia Tech, 1 Duckpond Drive, Blacksburg, Virginia 24061-0442, USA.

Abstract

Signaling through neurotrophic receptors is necessary for differentiation and survival of the developing nervous system. The present study examined the effects of the organic mercury compound thimerosal on nerve growth factor signal transduction and cell death in a human neuroblastoma cell line (SH-SY5Y cells). Following exposure to 100 ng/ml NGF and increasing concentrations of thimerosal (1 nM-10 microM), we measured the activation of TrkA, MAPK, and PKC-delta. In controls, the activation of TrkA MAPK and PKC-delta peaked after 5 min of exposure to NGF and then decreased but was still detectable at 60 min. Concurrent exposure to increasing concentrations of thimerosal and NGF for 5 min resulted in a concentration-dependent decrease in TrkA and MAPK phosphorylation, which was evident at 50 nM for TrkA and 100 nM for MAPK. Cell viability was assessed by the LDH assay. Following 24-h exposure to increasing concentrations of thimerosal, the EC50 for cell death in the presence or absence of NGF was 596 nM and 38.7 nM, respectively. Following 48-h exposure to increasing concentrations of thimerosal, the EC50 for cell death in the presence and absence of NGF was 105 nM and 4.35 nM, respectively. This suggests that NGF provides protection against thimerosal cytotoxicity. To determine if apoptotic versus necrotic cell death was occurring, oligonucleosomal fragmented DNA was quantified by ELISA. Control levels of fragmented DNA were similar in both the presence and absence of NGF. With and without NGF, thimerosal caused elevated levels of fragmented DNA appearing at 0.01 microM (apoptosis) to decrease at concentrations >1 microM (necrosis). These data demonstrate that thimerosal could alter NGF-induced signaling in neurotrophin-treated cells at concentrations lower than those responsible for cell death.

PMID: 15843506 [PubMed - indexed for MEDLINE]Free Article

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Toxicol In Vitro. 2005 Mar;19(2):191-8.

Flow-cytometric analysis on cytotoxic effect of thimerosal, a preservative in vaccines, on lymphocytes dissociated from rat thymic glands.

Ueha-Ishibashi T, Oyama Y, Nakao H, Umebayashi C, Hirama S, Sakai Y, Ishida S, Okano Y.

Laboratory of Cellular Signaling, Faculty of Integrated Arts and Sciences, The University of Tokushima, Minami-Jyosanjima 1-1, Tokushima 770-8502, Japan.

Abstract

There is a concern on the part of public health community that adverse health consequence by thimerosal, a preservative in vaccines for infants, may occur among infants during immunization schedule. Therefore, the cytotoxic action of thimerosal was examined on lymphocytes dissociated from thymic glands of young rats using a flow cytometer and respective fluorescent probes for monitoring changes in intracellular Ca2+ concentration ([Ca2+]i) and membrane potential, and for discriminating intact living cells, apoptotic living cells and dead cells. Incubation with thimerosal at 3 microM or more (up to 30 microM) for 60 min depolarized the membranes, associated with increasing the [Ca2+]i. Thimerosal at 30 microM induced an apoptotic change in membranes of almost all living cells. Furthermore, the prolonged incubation with 30 microM thimerosal induced a loss of membrane integrity, leading to cell death. Since the blood concentration of thimerosal after receiving vaccines is theoretically submicromolar, it may be unlikely that thimerosal affects lymphocytes of infants.

PMID: 15649632 [PubMed - indexed for MEDLINE]

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Neurotoxicology. 2005 Jan;26(1):1-8.

Thimerosal neurotoxicity is associated with glutathione depletion: protection with glutathione precursors.

James SJ, Slikker W 3rd, Melnyk S, New E, Pogribna M, Jernigan S.

Department of Pediatrics, University of Arkansas for Medical Sciences and Arkansas Children's Hospital Research Institute, Little Rock, AR 72202, USA. jamesjill@uams.edu

Abstract

Thimerosol is an antiseptic containing 49.5% ethyl mercury that has been used for years as a preservative in many infant vaccines and in flu vaccines. Environmental methyl mercury has been shown to be highly neurotoxic, especially to the developing brain. Because mercury has a high affinity for thiol (sulfhydryl (-SH)) groups, the thiol-containing antioxidant, glutathione (GSH), provides the major intracellular defense against mercury-induced neurotoxicity. Cultured neuroblastoma cells were found to have lower levels of GSH and increased sensitivity to thimerosol toxicity compared to glioblastoma cells that have higher basal levels of intracellular GSH. Thimerosal-induced cytotoxicity was associated with depletion of intracellular GSH in both cell lines. Pretreatment with 100 microM glutathione ethyl ester or N-acetylcysteine (NAC), but not methionine, resulted in a significant increase in intracellular GSH in both cell types. Further, pretreatment of the cells with glutathione ethyl ester or NAC prevented cytotoxicity with exposure to 15 microM Thimerosal. Although Thimerosal has been recently removed from most children's vaccines, it is still present in flu vaccines given to pregnant women, the elderly, and to children in developing countries. The potential protective effect of GSH or NAC against mercury toxicity warrants further research as possible adjunct therapy to individuals still receiving Thimerosal-containing vaccinations.

PMID: 15527868 [PubMed - indexed for MEDLINE]


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Int J Mol Med. 2005 Dec;16(6):971-7.

Thimerosal induces neuronal cell apoptosis by causing cytochrome c and apoptosis-inducing factor release from mitochondria.

Yel L, Brown LE, Su K, Gollapudi S, Gupta S.

Department of Medicine, University of California, Irvine, CA 92697, USA. lyel@uci.edu

Abstract

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.

PMID: 16273274 [PubMed - indexed for MEDLINE]
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