Dish of the Day: Red Herrings stuffed with Thimerosal - guaranteed 49.5% Mercury. Lisa Blakemore Brown 5th March 2005 British Medical Journal

Dish of the Day: Red Herrings stuffed with Thiomersal - guaranteed 49.5% Mercury.
5 March 2005

Lisa C Blakemore-Brown, Psychologist UK based
Send response to journal: Re: Dish of the Day: Red Herrings stuffed with Thiomersal - guaranteed 49.5% Mercury.
Thiomersal/Thimerosal, a preservative developed in the late 1920's was manufactured by Eli Lilly and has been routinely used in vaccines and many other products since that time.
Thiomersal/Thimerosal contains 49.5% ethyl mercury. Methyl mercury is known to accumulate in fish and for this reason there is a safe daily limit for the amount of mercury adults are advised to not exceed. This is 0.1 mcg of Methyl Mercury per kilogram of weight. Babies have been given 25 microgrammes in each of the following vaccines: whole cell DTP and Hib, and the Hep B in the US. During this same period many very small premature babies have survived whereas in the past they would not have done so.
Over the course of the last century, individuals were given single vaccines with single amounts of mercury, but with the introduction of triple vaccines the amount of mercury contained within the preservative was multiplied and the cumulative effects are only just now being discovered by the public. Early papers on autism and battered baby syndrome appeared following the introduction of vaccines containing this preservative in the 30's and 40's and following the introduction of triple vaccines there has been an epidemic of autism - and genetic epidemics do not occur without some outside influence. It is also interesting that the Shaken Baby Syndrome and parent blame theories for odd medical presentations, have also vastly increased since these triple vaccines were brought in.
Thiomersal is still used in flu vaccines and other products but has been phased out in many childhood vaccines, but only very recently. For the UK it was August 2004 when the announcement was made.
Public Health agencies around the world have known about concerns with this neurotoxin for some years, and certainly since 1997 when the US Congress passed the FDA Modernization Act requiring the FDA to review all drugs that contained Mercury and determine their effect on humans. (3)
In 2000, recognising the cumulative amount of mercury in the triple vaccines, the FDA stated that children were receiving levels of mercury via this preservative far in excess of the 'safe' guidelines. Rough estimates suggested a child could be injected with 40 times the amount of mercury considered safe (3)
Despite the expected red herring arguments of the defensive, and the collaborative efforts of many countries to handle worrying data, many lab studies have explored the toxic effects of Thiomersal in humans and animals.
Evidence that Thiomersal can create changes at cellular level is rife in the literature (6,7,14,17,18.)A recent study concluded 'thimerosal is genotoxic in the cytochalasin B block micronucleus test with human lymphocytes. These data raise some concern on the widespread use of thimerosal.'(12)
Changes to phenotypes have been recognised (13) and the tapestry model allows for these various synergistic effects. For instance, it has been consistently shown that Thiomersal(Thimerosal) is a neurotoxin which is linked to the depletion of the protective anti-oxidant, glutathione (1,13,14,15,18,19)and that some individuals are more susceptible than others to such effects (4,16) Autistic individuals have been found to have lowered levels of glutathione and to have greater difficulties excreting mercury.
It is not impossible that earlier generations of constitutionally susceptible individuals were affected by the Mercury in their very early vaccinations, phenotypes were altered and in turn their offspring increased their risk factors for susceptibility to mercury damage.
Such susceptibilities, combined with an already weak immune system - in a baby. a medically vulnerable individual or an elderly person - further increase the risk factors.
As the changes triggered by Thiomersal appear at cellular level, including cell death and increased cellular permeability, it is not surprising that many forms of impairment are found.
'Exposure to organic mercury leads to primarily neurologic effects but a number of other organ systems may also be involved, including gastrointestinal, respiratory, hepatic, immune, dermal, and renal.' (2) Travis Haws has discussed Louis Reik's Disseminated Vasculomyelinopathy:an Immune Complex Disease relating to the multiple effects of infection once the immune system has been compromised. (20)
Effects of the preservative in preparations have found retinal (21) and corneal damage. (22)
The consistent reference to allergic effects and immune system interference (4, 5) maps onto my own findings that early vaccines seemed to trigger various allergies and intolerances in some children (See CASE ONE 2004 ebmj as an example taken directly from the medical notes)
My very early observations during the mid to late 1990's was that it was these medically vulnerable and allergic individuals who were more likely to have reacted to the MMR - which contains no Thiomersal. (23) Unfortunately because of Red Herring arguments and denial of what is known, we have yet to fully explore in a scientific way, why so many children reacted to vaccines.
In 2000 a meeting held in Simpsonwood US, revealed significant findings in a study by Verstraeten in relation to the emergence of a variety of neurodevelopmental disorders including tics, ADHD, speech and language and motor impairments - and Thiomersal. It has taken the Freedom of Information Act to expose what was shown in that meeting - and the steps taken to cover it up.
The epidemic of health and developmental problems in so called advanced countries is now undeniable. 1 in 10 children suffer from gastrointestinal disorders, 1 in 4 have asthma. A colleague last week mentioned that out of 16 children on a school trip involving his child, 15 had asthma. Children are arriving at school unable to use a knife and fork, pay attention or understand their social worlds, highly sensitised to all aspects of the real world.
When we have removed the Red Herrings - and those who cook them up - we might begin to take responsibility again for the health of millions across the world.
Refs:
1: 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
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.
2: Toxicol Ind Health. 2002 Apr;18(3):109-60.
Organic mercury compounds: human exposure and its relevance to public health.
Risher JF, Murray HE, Prince GR.
Agency for Toxic Substances and Disease Registry, Division of Toxicology, Toxicology Information Branch, Clifton Road, Atlanta, Georgia 30333, USA.
Humans may be exposed to organic forms of mercury by either inhalation, oral, or dermal routes, and the effects of such exposure depend upon both the type of mercury to which exposed and the magnitude of the exposure. In general, the effects of exposure to organic mercury are primarily neurologic, while a host of other organ systems may also be involved, including gastrointestinal, respiratory, hepatic, immune, dermal, and renal. While the primary source of exposure to organic mercury for most populations is the consumption of methylmercury-contaminated fish and shellfish, there are a number of other organomercurials to which humans might be exposed. The antibacterial and antifungal properties of organomercurials have resulted in their long use as topical disinfectants (thimerosal and merbromin) and preservatives in medical preparations (thimerosal) and grain products (both methyl and ethyl mercurials). Phenylmercury has been used in the past in paints, and dialkyl mercurials are still used in some industrial processes and in the calibration of certain analytical laboratory equipment. The effects of exposure to different organic mercurials by different routes of exposure are summarized in this article.
3. Trade Off: Vaccine Maker Profits and Autism. Evelyn Pringle . Independent News Media. 4th March 2005
4: Med Pr. 2001;52(1):45-51.
[Genetic polymorphism of glutathione s-transferase as a factor predisposing to allergic dermatitis]
[Article in Polish]
Lutz W, Tarkowski M, Nowakowska E.
Zakladu Diagnostyki Laboratoryjnej, Instytutu Medycyny Pracy, Lodzi.
In the inductive phase of contact allergic dermatitis, simple chemical compounds (haptens) produce together with epidermic proteins adducts presented by Langerhans cells to T lymphocytes. Binding to protein carrier is a necessary condition of transforming a low-molecular allergen into immunogenic one and evoking immunological reaction. The production of allergen adducts with proteins is conditioned by the presence of electrophilic groups in their molecules, or their acquiring during biotransformation phase I. Active allergen metabolites undergo further alterations during biotransformation phase II which leads most frequently to the decline in their chemical activity and more rapid excretion from the body. The number of reactive metabolites (reactive allergens) available for producing adducts with proteins keeps the balance between activation and deactivation reactions. Glutathione S-transferases play a particular role in the allergens (or their metabolites) deactivation process in biotransformation phase II. These enzymes catalyse reactions responsible for the declined electrophilic potential of allergens (or their metabolites), and thus for the decrease in the number of allergen molecules able to produce protein covalent bindings (adducts). Glutathione S-transferases, occurring in the human cellular cytoplasm belong to five classes: alpha(GST A), mu(GST M), theta(GST P), pi(GST T) and Z(GST Z), as well as to one class present in microsomes. The study indicated the presence of isoenzymes GST T1 and GST M1 in the skin. Both isoforms participate in the process of low-molecular allergen biotransformation. Carriers of defective genes GST T1 and/or GST M1 are more vulnerable to allergenic effect of some allergens, e.g. thimerosal, which is associated with the absence of or decrease in the activity of isoenzymes GST T1 and GST M1.
5: Cell Biol Toxicol. 1999 Feb;15(1):57-62.
Mechanisms of drug-induced allergic contact dermatitis.
Lebrec H, Bachot N, Gaspard I, Kerdine S, Guinnepain MT, Laurent J, Pallardy M.
INSERM U 461, Faculte de Pharmacie Paris Sud, Chatenay-Malabry, France. hlebrec@infobiogen.fr
Allergic contact dermatitis is induced by a wide variety of drugs that trigger specific immune responses following topical exposure. Identified chemical structures involved in such reactions include the mercuric and thiosalicylic acid groups of thimerosal, the diphenylketone group of the anti- inflammatory drug ketoprofen, the amide or ester structure of local anesthetics, and the side-chain and thiazolidine ring of beta-lactams. The T cell responses to such compounds involve CD4+ and CD8+ alphabeta+ T lymphocytes and also CD4 /CD8 gammadelta+ T cells. Although "T helper 2" cytokine production by drug- specific human T cells from patients with allergic contact dermatitis has been described, T helper 1-like and T cytotoxic 1-like responses clearly play key roles in this cutaneous reaction.
6. Int J Biochem. 1994 Jan;26(1):93-6.
Effect of thimerosal on cytosolic calcium and phosphatidylserine synthesis in Jurkat T cells.
Pelassy C, Breittmayer JP, Ticchioni M, Aussel C.
INSERM U343, Faculte de Medecine, Nice, France.
1. We investigated the effect of the thiol reagent, thimerosal on calcium movements in the Jurkat T cell line. 2. Thimerosal induced a rise in cytosolic Ca2+ concentration due both to a release of Ca2+ from intracellular stores and a Ca2+ influx. 3. Thimerosal, released Ca2+ from the same intracellular stores than CD3 mAb and ionomycin. 4. Emptying the Ca2+ intracellular stores was accompanied by a marked decrease of phosphatidylserine synthesis indicating that phosphatidylserine synthesis occurs within or close to the endoplasmic reticulum Ca(2+)-stores as previously described in CD3-, ionomycin- or Ca(2+)-ATPase inhibitor-treated lymphocytes.
7. Biochim Biophys Acta. 1992 Oct 19;1111(1):65-74.
Effect of the sulfhydryl reagent thimerosal on cytosolic free Ca2+ and membrane potential of thymocytes.
Gukovskaya AS, Trepakova ES, Zinchenko VP, Korystov YN, Bezuglov VV.
Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, Moscow Region.
The sulfhydryl reagent thimerosal at concentrations 5-100 microM has been found to induce a variety of changes in ion transport in rat thymocytes. In particular, [Ca2+]i increases about 10-fold from the basal level. The [Ca2+]i response to thimerosal displays a two-stage time course, with the main [Ca2+]i rise during the second stage. Evidence has been obtained for the depletion of intracellular Ca2+ pools in thimerosal-treated cells, however, Ca2+ mobilization from intracellular stores does not contribute significantly into [Ca2+]i rise. Thimerosal elicits permeability not only for Ca2+, but also for Mn2+ and Ni2+, which is Ca(2+)-dependent. We failed to get any evidence on thimerosal- induced inhibition of the plasma membrane Ca(2+)-ATPase. The induction of Ca2+ influx, rather than inhibition of Ca(2+)-ATPase, accounts for the disturbance of [Ca2+]i homeostasis in thimerosal-treated cells. Thimerosal also elicits changes in monovalent ion fluxes resulting in marked depolarization. The latter seems unrelated to the changes in [Ca2+]i and is suggested to be mediated both by increased permeability for Na+ and a decreased one for K+. Thimerosal significantly stimulates AA release from thymocytes. Evidence has been presented that AA metabolite(s), probably, LO product(s), may mediate the changes in the transport of mono- and divalent cations elicited by the sulfhydryl reagent. Prolonged treatment of thymocytes with thimerosal resulted in cell death.
8. Lancet. 1991 Aug 3;338(8762):315-6.
Ecotoxicol Environ Saf. 1985 Oct;10(2):150-8.
Effect of organomercurial poisoning on the peripheral blood and metabolite levels of a freshwater fish.
Gill TS, Pant JC.
This work evaluated the hematological and biochemical changes in the fish, Puntius conchonius, under experimental organomercurial poisoning. Long- term (8 weeks) exposure to 3.63 and 6.03 mg/liter methoxyethyl mercuric chloride (MEMC) (0.2 and 0.33 fractions of 96-hr LC50) led to morphological aberrations in mature erythrocytes including nuclear and cytoplasmic deterioration, vacuolation, chromatin condensation, and hypochromia. Immature erythrocytes showing membrane leakage were also encountered. Erythrocyte count and hemoglobin (Hb) were significantly lowered after 1 and 3 weeks followed by a marginal rise persisting upto 8 weeks. Differential leucocyte counts revealed significant thrombocytopenia, lymphocytosis, and neutropenia. Collateral evaluation of blood glucose and tissue glycogen levels revealed significant hyperglycemia as well as glycogen depletion in liver and brain. Heart glycogen content evinced a substantial increase after 5 and 8 weeks exposure.
9. Rev Neurol (Paris). 1979;135(11):827-33.
[Morvan's fibrillary chorea]
[Article in French]
de Bray JM, Emile J, Basle M, Morer T, Bastard J.
Two cases of Morvan's chorea are reported. One of the patients presented the characteristic of having had two attacks, the first after organic mercury preparations, and the second after gold salts for inflammatory rheumatism. The second case had facial fibrillations only, and this was followed by a regressive polyradiculoneuritis one month later. This latter case raises certain diagnostic problems. The existence of a particular type of immuno-allergic tendency could be validly related to a triggering effect of various etiological agents (metals such as mercury or gold salts, or infective agents). The absence of hypotonia, and a regressive course appear to be the characteristics that distinguish fibrillary chorea from the continuous activity syndrome of the muscle fibers described by Isaacs.
10. Contact Dermatitis. 1980 Jun;6(4):241-5.
Merthiolate hypersensitivity and vaccination.
Forstrom L, Hannuksela M, Kousa M, Lehmuskallio E.
Epicutaneous tests with 0.1% merthiolate in petrolatum showed hypersensitivity in 96 of 4647 eczema patients (2.0%) and in seven of 105 healthy recruits (7%). There was a marked preponderance of young age classes in the eczema group. Twelve of 41 merthiolate-positive patients tested reacted to mercury alone, three to thiosalicylic acid alone and one to both. The remaining 25 patients reacted to neither of the individual components although the merthiolate complex as a whole gave a positive test result. Forty-five of the merthiolate- positive patients were tested subcutaneously with 0.5 ml of a 0.01% merthiolate solution, i.e. a dose equal to that contained in one shot of tetanus toxoid, for example. Nine patients developed a local reaction at the site of the injection, and the area became eczematous in four cases. In one of the patients the eczema spread over the body, causing fever. Since merthiolate-sensitive patients also react to merthiolate administered intracutaneously, the vaccinator should avoid the use of a needle whose outer surface has been contaminated when the vaccine was aspirated from the bottle. However, even when this precautionary measure is taken, local reactions can be expected in such a high percentage of merthiolate-sensitive persons that merthiolate in vaccines should be replaced by another antibacterial agent.
11. Contact Dermatitis. 1986 Nov;15(5):309-10.
Reactions to merthiolate in infants.
Novak M, Kvicalova E, Friedlanderova B.
12. Arch Toxicol. 2003 Jan;77(1):50-5. Epub 2002 Nov 06.
Thimerosal induces micronuclei in the cytochalasin B block micronucleus test with human lymphocytes.
Westphal GA, Asgari S, Schulz TG, Bunger J, Muller M, Hallier E.
Department of Occupational Health, Georg-August-University Gottingen, Waldweg 37, 37073 Gottingen, Germany. gwestph@gwdg.de
Thimerosal is a widely used preservative in health care products, especially in vaccines. Due to possible adverse health effects, investigations on its metabolism and toxicity are urgently needed. An in vivo study on chronic toxicity of thimerosal in rats was inconclusive and reports on genotoxic effects in various in vitro systems were contradictory. Therefore, we reinvestigated thimerosal in the cytochalasin B block micronucleus test. Glutathione S-transferases were proposed to be involved in the detoxification of thimerosal or its decomposition products. Since the outcome of genotoxicity studies can be dependent on the metabolic competence of the cells used, we were additionally interested whether polymorphisms of glutathione S-transferases (GSTM1, GSTT1, or GSTP1) may influence the results of the micronucleus test with primary human lymphocytes. Blood samples of six healthy donors of different glutathione S-transferase genotypes were included in the study. At least two independent experiments were performed for each blood donor. Significant induction of micronuclei was seen at concentrations between 0.05-0.5 micro g/ml in 14 out of 16 experiments. Thus, genotoxic effects were seen even at concentrations which can occur at the injection site. Toxicity and toxicity-related elevation of micronuclei was seen at and above 0.6 micro g/ml thimerosal. Marked individual and intraindividual variations in the in vitro response to thimerosal among the different blood donors occurred. However, there was no association observed with any of the glutathione S-transferase polymorphism investigated. In conclusion, thimerosal is genotoxic in the cytochalasin B block micronucleus test with human lymphocytes. These data raise some concern on the widespread use of thimerosal.
13. Int J Hyg Environ Health. 2001 Jul;203(5-6):479-81.
Inhibition of the human erythrocytic glutathione-S-transferase T1 (GST T1) by thimerosal.
Muller M, Westphal G, Vesper A, Bunger J, Hallier E.
Department of Occupational and Social Medicine, Georg-August- University Gottingen, D-37073 Gottingen, Germany. mmuelle3@gwdg.de
We have investigated the interaction of thimerosal, a widely used antiseptic and preservative, with the human erythrocytic GST T1 (glutathione-S- transferase T1). This detoxifying enzyme is expressed in the erythrocytes of solely the human species and it displays a genetic polymorphism. Due to this polymorphism about 25% of the individuals of the caucasian population lack this activity ("non-conjugators"), while 75% show it ("conjugators") (Hallier, E., et al., 1993). Using our newly developed HPLC-fluorescence detection assay (Muller, M., et al., 2001) we have profiled the kinetics of enzyme inhibition in erythrocyte lysates of two individuals previously identified as "normal conjugator" (medium enzyme activity) and "super-conjugator" (very high activity). For the normal conjugator we have determined a 2.77 mM thimerosal concentration to inhibit 50% of the GST T1 activity. In the case of the super-conjugator a 2.3 mM thimerosal concentration causes a 50% inhibition of the enzyme activity. For both phenotypes a 14.8 mM thimerosal concentration results in residual enzyme activities equal to those typically detected in non-conjugator lysates. Thus, sufficiently high doses of thimerosal may be able to change the phenotypic status of an individual--at least in vitro--by inhibition of the GST T1 enzyme.
14. Int Arch Occup Environ Health. 2000 Aug;73(6):384-8.
Homozygous gene deletions of the glutathione S-transferases M1 and T1 are associated with thimerosal sensitization.
Westphal GA, Schnuch A, Schulz TG, Reich K, Aberer W, Brasch J, Koch P, Wessbecher R, Szliska C, Bauer A, Hallier E.
Abteilung fur Arbeits- und Sozialmedizin, Georg-August-Universitat Gottingen, Germany. gwestph@gwdg.de
OBJECTIVE: Thimerosal is an important preservative in vaccines and ophthalmologic preparations. The substance is known to be a type IV sensitizing agent. High sensitization rates were observed in contact-allergic patients and in health care workers who had been exposed to thimerosal-preserved vaccines. There is evidence for the involvement of the glutathione system in the metabolism of thimerosal or its decomposition products (organomercury alkyl compounds). Thus detoxification by polymorphically expressed glutathione S-transferases such as GSTT1 and GSTM1 might have a protective effect against sensitization by these substances. METHODS: To address this question, a case control study was conducted, including 91 Central European individuals with a positive patch-test reaction to thimerosal. This population was compared with 169 healthy controls and additionally with 114 individuals affected by an allergy against para-substituted aryl compounds. The latter population was included in order to test whether possible associations were due to substance-specific effects, or were a general feature connected with type IV immunological diseases. Homozygous deletions of GSTT1 and GSTM1 were determined by polymerase chain reaction. RESULTS: Glutathione S-transferase M1 deficiency was significantly more frequent among patients sensitized to thimerosal (65.9%, P = 0.013) compared with the healthy control group (49.1%) and the "para-compound" group (48%, P = 0.034). Glutathione S-transferase T1 deficiency in the thimerosal/mercury group (19.8%) was barely elevated versus healthy controls (16.0%) and the "para-compound" group (14.0%). The combined deletion (GSTT1-/GSTM1-) was markedly more frequent among thimerosal-sensitized patients than in healthy controls (17.6% vs. 6.5%, P = 0.0093) and in the "para- compound" group (17.6% vs. 6.1%, P =0.014), revealing a synergistic effect of these enzyme deficiencies (healthy controls vs. thimerosal GSTM1 negative individuals, OR = 2.0 [CI = 1.2-3.4], GSTT1-, OR = 1.2 [CI = 0.70-2.1], GSTM1/T1-, OR = 3.1 [CI = 1.4-6.5]). CONCLUSIONS: Since the glutathione-dependent system was repeatedly shown to be involved in the metabolism of thimerosal decomposition products, the observed association may be of functional relevance.
PMID: 11007341 [PubMed - indexed for MEDLINE]
15 Toxicol In Vitro. 2004 Oct;18(5):563-9.
Property of thimerosal-induced decrease in cellular content of glutathione in rat thymocytes: a flow cytometric study with 5-chloromethylfluorescein diacetate.
Ueha-Ishibashi T, Tatsuishi T, Iwase K, Nakao H, Umebayashi C, Nishizaki Y, Nishimura Y, Oyama Y, Hirama 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.
There is a concern on the part of public health community that adverse health consequences by thimerosal, a preservative in vaccines for infants, may occur among infants during immunization schedule. Therefore, the effect of thimerosal on cellular content of glutathione was examined on thymocytes obtained from 4-week-old rats using a flow cytometer and 5-chloromethylfluorescein diacetate. Thimerosal at concentrations ranging from 1 to 10 microM reduced the cellular content of glutathione in a concentration-dependent manner, and the complete depletion of cellular glutathione was observed when the cells were treated with 30 microM thimerosal. L-Cysteine significantly attenuated the actions of thimerosal to reduce the glutathione content and to increase the intracellular Ca2+ concentration. Prolonged incubation (24 h) with 1-3 microM thimerosal induced the apoptosis. The cytotoxic action of thimerosal was greatly augmented when the cells suffered oxidative stress induced by H2O2. It may be unlikely that thimerosal exerts potent cytotoxic action under the in vivo condition because the blood concentration of thimerosal after receiving vaccines does not seem to reach micromolar range and nonprotein thiols at micromolar concentrations are present in the blood.
16: J Invest Dermatol. 2003 Nov;121(5):1039-44.
Thiol antioxidants block the activation of antigen-presenting cells by contact sensitizers.
Bruchhausen S, Zahn S, Valk E, Knop J, Becker D.
Department of Dermatology, University of Mainz, Mainz, Germany.
Strong contact sensitizers are able to induce signal transduction mechanisms such as tyrosine phosphorylation and activation of MAP kinases in antigen-presenting cells. We studied the capacity of different antioxidants (ascorbic acid, alpha-tocopherol, pyrrolidine dithiocarbamate, N- acetylcysteine, and glutathione) to block the increase in tyrosine phosphorylation in human monocytes seen after stimulation with strong contact sensitizers. Human peripheral blood mononuclear cells were stimulated with 5-chloro-2-methylisothiazolinone plus 2-methylisothiazolinone in the presence or absence of these antioxidants. The total amount of membrane-associated phosphotyrosine in CD14+ cells was quantified using flow cytometric techniques. Complete inhibition of tyrosine phosphorylation was noticed when cells were stimulated in the presence of N-acetylcysteine or glutathione. Using N-acetylcysteine as inhibitor similar results were obtained for cells stimulated with formaldehyde, thimerosal methyldibromoglutaronitrile, diphenylcyclopropenone, p-phenylenediamine, toluene-2,5-diamine, and 2,4-dinitrofluorobenzene. By use of a trinitrophenyl-specific monoclonal antibody it was shown that N-acetylcysteine as well as cysteine prevents the binding of 2,4,6-trinitrochlorobenzene to proteins in monocytes and monocyte-derived mature dendritic cells. Furthermore, the capacity of N-acetylcysteine to block the activation of p38 and ERK1/2 MAP kinases by 2,4,6-trinitrochlorobenzene was demonstrated. The radical scavengers ascorbic acid and alpha-tocopherol as well as the nuclear factor kappaB inhibitor pyrrolidine dithiocarbamate failed to prevent the increase in tyrosine phosphorylation. Our data present evidence that reactive oxygen species as well as transcription factor nuclear factor kappaB seem to be unimportant for the induction of tyrosine phosphorylation by contact sensitizers. On the other hand, protection of thiol groups using compounds with free sulfhydryl groups is very effective to block this process. This finding may have implications for prevention of occupational sensitization to strong contact allergens.
17. Toxicology. 2004 Jan 15;195(1):77-84.
Effect of thimerosal, a preservative in vaccines, on intracellular Ca2+ concentration of rat cerebellar neurons.
Ueha-Ishibashi T, Oyama Y, Nakao H, Umebayashi C, Nishizaki Y, Tatsuishi T, Iwase K, Murao K, Seo H.
Laboratory of Cellular Signaling, Faculty of Integrated Arts and Sciences, The University of Tokushima, Tokushima 770-8502, Japan.
The effect of thimerosal, an organomercurial preservative in vaccines, on cerebellar neurons dissociated from 2-week-old rats was compared with those of methylmercury using a flow cytometer with appropriate fluorescent dyes. Thimerosal and methylmercury at concentrations ranging from 0.3 to 10 microM increased the intracellular concentration of Ca2+ ([Ca2+]i) in a concentration-dependent manner. The potency of 10 microM thimerosal to increase the [Ca2+]i was less than that of 10 microM methylmercury. Their effects on the [Ca2+]i were greatly attenuated, but not completely suppressed, under external Ca(2+)-free condition, suggesting a possibility that both agents increase membrane Ca2+ permeability and release Ca2+ from intracellular calcium stores. The effect of 10 microM thimerosal was not affected by simultaneous application of 30 microM L-cysteine whereas that of 10 microM methylmercury was significantly suppressed. The potency of thimerosal was similar to that of methylmercury in the presence of L-cysteine. Both agents at 1 microM or more similarly decreased the cellular content of glutathione in a concentration-dependent manner, suggesting an increase in oxidative stress. Results indicate that thimerosal exerts some cytotoxic actions on cerebellar granule neurons dissociated from 2-week-old rats and its potency is almost similar to that of methylmercury.
18: Genes Immun. 2002 Aug;3(5):270-8.
Biochemical and molecular basis of thimerosal-induced apoptosis in T cells: a major role of mitochondrial pathway.
Makani S, Gollapudi S, Yel L, Chiplunkar S, Gupta S.
Cellular and Molecular Immunology Laboratories, Division of Basic and Clinical Immunology, University of California, Irvine 92697, USA.
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.
19. Int Arch Occup Environ Health. 2000 Aug;73(6):384-8.
Homozygous gene deletions of the glutathione S-transferases M1 and T1 are associated with thimerosal sensitization.
Westphal GA, Schnuch A, Schulz TG, Reich K, Aberer W, Brasch J, Koch P, Wessbecher R, Szliska C, Bauer A, Hallier E.
Abteilung fur Arbeits- und Sozialmedizin, Georg-August-Universitat Gottingen, Germany. gwestph@gwdg.de
OBJECTIVE: Thimerosal is an important preservative in vaccines and ophthalmologic preparations. The substance is known to be a type IV sensitizing agent. High sensitization rates were observed in contact-allergic patients and in health care workers who had been exposed to thimerosal-preserved vaccines. There is evidence for the involvement of the glutathione system in the metabolism of thimerosal or its decomposition products (organomercury alkyl compounds). Thus detoxification by polymorphically expressed glutathione S-transferases such as GSTT1 and GSTM1 might have a protective effect against sensitization by these substances. METHODS: To address this question, a case control study was conducted, including 91 Central European individuals with a positive patch-test reaction to thimerosal. This population was compared with 169 healthy controls and additionally with 114 individuals affected by an allergy against para-substituted aryl compounds. The latter population was included in order to test whether possible associations were due to substance-specific effects, or were a general feature connected with type IV immunological diseases. Homozygous deletions of GSTT1 and GSTM1 were determined by polymerase chain reaction. RESULTS: Glutathione S-transferase M1 deficiency was significantly more frequent among patients sensitized to thimerosal (65.9%, P = 0.013) compared with the healthy control group (49.1%) and the "para-compound" group (48%, P = 0.034). Glutathione S-transferase T1 deficiency in the thimerosal/mercury group (19.8%) was barely elevated versus healthy controls (16.0%) and the "para-compound" group (14.0%). The combined deletion (GSTT1-/GSTM1-) was markedly more frequent among thimerosal-sensitized patients than in healthy controls (17.6% vs. 6.5%, P = 0.0093) and in the "para- compound" group (17.6% vs. 6.1%, P =0.014), revealing a synergistic effect of these enzyme deficiencies (healthy controls vs. thimerosal GSTM1 negative individuals, OR = 2.0 [CI = 1.2-3.4], GSTT1-, OR = 1.2 [CI = 0.70-2.1], GSTM1/T1-, OR = 3.1 [CI = 1.4-6.5]). CONCLUSIONS: Since the glutathione-dependent system was repeatedly shown to be involved in the metabolism of thimerosal decomposition products, the observed association may be of functional relevance.
20. Travis Haws Re:Re:Re:Re: A Fatal Misdiagnosis. bmj. February 18th 2005.
21, J Neurosci Res. 1996 Apr 15;44(2):149-56.
Pharmacological characterization of inositol-1,4,5,-trisphosphate binding to membranes from retina and retinal cultures.
Lopez-Colome AM, Lee I.
Instituto de Fisiologia Celular, Universidad Nacional Autonoma de Mexico.
Light and excitatory amino acids (EAA) stimulate the phosphoinositide cycle in the vertebrate retina. The regulation of Ca2+ release from intracellular stores by inositol-1,4, 5-trisphosphate (IP3) involves an interaction of this compound with specific receptors. By means of [3H]IP3-specific binding, we studied the kinetic and pharmacological properties of IP3 receptors in the chick retina as well as in primary cultures of neurons and glia from this tissue. The equilibrium time for the binding reaction was 15 min and was optimal at alkaline pH (8.3). IP3 receptor displayed high affinity (K(B) approximately 40 nM) and selectivity for D-IP3, compared to D-IP4 > L-IP3 > D-IP2 > D- IP1. These characteristics were the same in subcellular fractions from outer (P1) and thinner (P2) plexiform layers, binding sites being more abundant in P2 (2.65 pmol/mg protein). IP3 receptors were present in both neuronal and glial cultures, but were concentrated in neuronal cultures. Binding was not affected by ryanodine, or caffeine, related to calcium-induced calcium release (CICR)channels, nor by the endoplasmic reticulum Ca2+ ATPase inhibitor thapsigargin, while heparin affectively inhibited IP3 binding. GSSG and thimerosal increased the affinity of [3H]IP3 binding from IC50 approximately 80 nM to IC50 approximately 40 nM; this effect was reversed by DTT. Binding in zero Ca2+ was decreased by low concentrations of Ca2+ (350 nM). These results suggest that actions of IP3 in the retina are regulated by physiological changes in intracellular pH and Ca2+ concentrations, as well as by the oxidation state of the receptor. Additionally, the presence of IP3 receptors in Muller glia opens the possibility of IP3 participation in nonsynaptic signalling through Ca2+ waves in glial cells.
22. Invest Ophthalmol Vis Sci. 1977 Apr;16(4):273-80.
Effect of the ophthalmic preservative thimerosal on rabbit and human corneal endothelium.
Van Horn DL, Edelhauser HF, Prodanovich G, Eiferman R, Pederson HF.
Widespread use of the mercurial-containing preservative thimerosal as an antibacterial agent in ophthalmic drugs and solutions warranted an investigation into its possible cytotoxic effects on the functional and ultrastructural integrity of the corneal endothelium. No changes in corneal thickness were observed during 5 hours' perfusion of the endothelium of rabbit and human corneas with 0.0001 and 0.0005 percent thimerosal in glutathione bicarbonate Ringer's solution (GBR). Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) of the endothelium of the 0.0001 percent group revealed normal ultrastructure. SEM and TEM of the endothelium of corneas perfused with 0.0005 percent thimerosal for 5 hours revealed condensed mitochondria, cytoplasmic vacuoles, and cytoplasmic flaps at the apical end of the cellular junctions. Perfusion of higher concentrations (0.001 and 0.005 perecnt) of thimerosal in GBR resulted in increases in corneal thickness after 2 hours and irreversible ultrastructural damage to the endothelial cells by 5 hours. Corneas perfused with 0.01 and 0.1 percent thimerosal in GBR showed a rapid and immediate increase in corneal thickness and endothelial cell death and necrosis within 1 hour. It is postulated that the mercury in thimerosal becomes bound to the cell membrane protein sulfhydryl groups, causing an increase in cellular permeability; These results suggest that the prolonged exposure of the corneal endothelium to thimerosal in the accepted antimicrobial dosage of 0.005 to 0.001 percent may result in functional and structural damage to the endothelium.
23. Blakemore-Brown Reweaving the Autistic Tapestry. Jessica Kingsley Publishers London 2002.
Competing interests: Expert in Autism and associated tapestry impairments