Suppression by Thimerosal of Ex-Vivo CD4+ T Cell Response to Influenza Vaccine and Induction of Apoptosis in Primary Memory T Cells

Thimerosal is a preservative used widely in vaccine formulations to prevent bacterial and fungal contamination in multidose vials of vaccine. Thimerosal was included in the multidose non-adjuvanted pandemic 2009 H1N1 vaccine Panenza. In the context of the analysis of the ex-vivo T cell responses directed against influenza vaccine, we discovered the in vitro toxicity Panenza, due to its content in thimerosal. Because thimerosal may skew the immune response to vaccines, we investigated in detail the ex-vivo effects of thimerosal on the fate and functions of T cells in response to TCR ligation. We report that ex-vivo exposure of quiescent or TCR-activated primary human T cells to thimerosal induced a dose-dependent apoptotic cell death associated with depolarization of mitochondrial membrane, generation of reactive oxygen species, cytochrome c release from the mitochondria and caspase-3 activation. Moreover, exposure to non-toxic concentrations of thimerosal induced cell cycle arrest in G0/G1 phase of TCR-activated T cells, and inhibition of the release of proinflammatory cytokines such as IFN gamma, IL-1 beta, TNF alpha, IL-2, as well as the chemokine MCP1. No shift towards Th2 or Th17 cells was detected. Overall these results underline the proapoptotic effect of thimerosal on primary human lymphocytes at concentrations 100 times less to those contained in the multidose vaccine, and they reveal the inhibitory effect of this preservative on T-cell proliferation and functions at nanomolar concentrations.     

In vitro study of thimerosal reactions in human whole blood and plasma surrogate samples

Because of its bactericidal and fungicidal properties, thimerosal is used as a preservative in drugs and vaccines and is thus deliberately injected into the human body. In aqueous environment, it decomposes into thiosalicylic acid and the ethylmercury cation. This organomercury fragment is a potent neurotoxin and is suspected to have similar toxicity and bioavailability like the methylmercury cation. In this work, human whole blood and physiological simulation solutions were incubated with thimerosal to investigate its behaviour and binding partners in the blood stream. Inductively coupled plasma with optical emission spectrometry (ICP-OES) was used for total mercury determination in different blood fractions, while liquid chromatography (LC) coupled to electrospray ionisation time-of-flight (ESI-TOF) and inductively coupled plasma-mass spectrometry (ICP-MS) provided information on the individual mercury species in plasma surrogate samples. Analogous behaviour of methylmercury and ethylmercury species in human blood was shown and an ethylmercury-glutathione adduct was identified.     

Are toxic biometals destroying your children’s future?

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.     

In vitro uptake of glutamate in GLAST-and GLT-1-transfected mutant CHO-K1 cells is inhibited by the ethylmercury-containing preservative thimerosal

Thimerosal, also known as thimersal, Merthrolate, or sodiumethyl-mercurithiosalicylate, is an organic mercurial compound that is used in a variety of commercial as well as biomedical applications. As a preservative, it is used in a number of vaccines and pharmaceutical products. Its active ingredient is ethylmercury. Both inorganic and organic mercurials are known to interfere with glutamate homeostasis. Brain glutamate is removed mainly by astrocytes from the extracellular fluid via high-affinity astroglial Na⁺-dependent excitatory amino acid transporters, glutamate/ aspartats transporter (GLAST) and glutamate transporter-1 (GLT-1). The effects of thimerosal on glutamate homeostasis have yet to be determined. As a first step in this process, we examined the effects of thimerosal on the transport of [³H]-D-aspartate, a nonmetabolizable glutamate analog, in Chinese hamster ovary (CHO) cells transfected with two glutamate transporter subtypes, GLAST (EAAT1) and GLT-1 (EAAT2). Additionally, studies were undertaken to determine the effects of thimerosal on mRNA and protein levels of these transporters. The results indicate that thimerosal treatment caused significant but selective changes in both glutamate transporter mRNA and protein expression in CHO cells. Thimerosal-mediated inhibition of glutamate transport in the CHO-K1 cell line DdB7 was more pronounced in the GLT-1-transfected cells compared with the GLAST-transfected cells. These studies suggest that thimerosal accumulation in the central nervous system might contribute to dysregulation of glutamate homeostasis.     

A study on the potential reprotoxic effects of thimerosal in male albino rats

Thimerosal is ethyl mercury based compound which is being used as a preservative in vaccines since decades. Pharmaceutical products and vaccines that contain thimerosal are among the potential source of mercury exposure. Current research was intended to ascertain the reprotoxic effects of thimerosal on rat testes. Twenty-four adult male albino rats were sorted into four groups (n = 6). The first group was a control group. Rats of experimental Group 2, 3 and 4 were treated with various dosages of thimerosal (0.5, 10, 50 mg/kg) respectively. Rats were decapitated after thirty days of trial and different parameters were analyzed. Thimerosal exposure resulted in a significant decrease in antioxidant enzyme activities including catalase (CAT), peroxidase (POD), superoxide dismutase (SOD), glutathione reductase (GSR) and increased levels of thiobarbituric acid reactive substances (TBARS). Different doses of thimerosal significantly decreased (p < 0.05) the concentration of plasma testosterone, luteinizing hormone (LH) and follicle stimulating hormone (FSH). Additionally, Daily sperm production (DSP) and efficiency of daily sperm production were significantly reduced followed by thimerosal exposure. Moreover, thimerosal significantly (p < 0.05) decreased the primary spermatocytes, secondary spermatocytes, number of spermatogonia along with spermatids. Thimerosal induced adverse histopathological and morphological changes in testicular tissues such as decreased Leydig cells, diameter of seminiferous tubules, tunica albuginea height and epithelial height. On the other hand, the increase in tubular lumen and interstitial spaces was observed due to thimerosal. These outcomes indicated that thimerosal has potential reprotoxic effects in male albino rats.     

Administration of Thimerosal to Infant Rats Increases Overflow of Glutamate and Aspartate in the Prefrontal Cortex: Protective Role of Dehydroepiandrosterone Sulfate

Thimerosal, a mercury-containing vaccine preservative, is a suspected factor in the etiology of neurodevelopmental disorders. We previously showed that its administration to infant rats causes behavioral, neurochemical and neuropathological abnormalities similar to those present in autism. Here we examined, using microdialysis, the effect of thimerosal on extracellular levels of neuroactive amino acids in the rat prefrontal cortex (PFC). Thimerosal administration (4 injections, i.m., 240 μg Hg/kg on postnatal days 7, 9, 11, 15) induced lasting changes in amino acid overflow: an increase of glutamate and aspartate accompanied by a decrease of glycine and alanine; measured 10–14 weeks after the injections. Four injections of thimerosal at a dose of 12.5 μg Hg/kg did not alter glutamate and aspartate concentrations at microdialysis time (but based on thimerosal pharmacokinetics, could have been effective soon after its injection). Application of thimerosal to the PFC in perfusion fluid evoked a rapid increase of glutamate overflow. Coadministration of the neurosteroid, dehydroepiandrosterone sulfate (DHEAS; 80 mg/kg; i.p.) prevented the thimerosal effect on glutamate and aspartate; the steroid alone had no influence on these amino acids. Coapplication of DHEAS with thimerosal in perfusion fluid also blocked the acute action of thimerosal on glutamate. In contrast, DHEAS alone reduced overflow of glycine and alanine, somewhat potentiating the thimerosal effect on these amino acids. Since excessive accumulation of extracellular glutamate is linked with excitotoxicity, our data imply that neonatal exposure to thimerosal-containing vaccines might induce excitotoxic brain injuries, leading to neurodevelopmental disorders. DHEAS may partially protect against mercurials-induced neurotoxicity.     

Mercury and autism: accelerating evidence?

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.     

Influence of free thiol group(s) on autoantibody-defined epitope of proliferating cell nuclear antigen.

Proliferating cell nuclear Ag (PCNA) is an intranuclear protein involved in DNA replication directed by DNA polymerase delta and is the target Ag of autoantibodies in some patients with SLE. There is evidence that the epitope on PCNA recognized by human autoantibodies is conformation-dependent and is not a continuous peptide sequence. Thimerosal, a mercury-containing sulfhydryl blocking compound, markedly reduced or abolished the reactivity of this autoantibody-defined PCNA epitope. The thimerosal effect was observed in various Ag-detecting systems including indirect immunofluorescence, immunodiffusion, immunoprecipitation, and flow cytometry. The mechanism of the thimerosal effect appeared to be mediated through free but not readily accessible sulfhydryl group or groups. The sulfhydryl-modification by thimerosal could be reversed by competition with thiol-containing compounds. Experimentally induced mAb to PCNA generated by immunization with purified PCNA have been shown to recognize epitopes that are continuous peptide sequences and these epitopes were not affected by thimerosal. It has been shown that the human autoantibody-defined epitope is related to the function of PCNA because autoantibodies are able to inhibit DNA polymerase delta directed DNA replication, whereas experimentally induced antibodies are not. These studies show that certain sulfhydryl groups in PCNA have a role in determining the antigenicity of the epitope recognized by autoantibody and raise the possibility that certain sulfhydryl groups might also be associated with its function.     

Neonatal Administration of Thimerosal Causes Persistent Changes in Mu Opioid Receptors in the Rat Brain

Thimerosal added to some pediatric vaccines is suspected in pathogenesis of several neurodevelopmental disorders. Our previous study showed that thimerosal administered to suckling rats causes persistent, endogenous opioid-mediated hypoalgesia. Here we examined, using immunohistochemical staining technique, the density of μ-opioid receptors (MORs) in the brains of rats, which in the second postnatal week received four i.m. injections of thimerosal at doses 12, 240, 1,440 or 3,000 μg Hg/kg. The periaqueductal gray, caudate putamen and hippocampus were examined. Thimerosal administration caused dose-dependent statistically significant increase in MOR densities in the periaqueductal gray and caudate putamen, but decrease in the dentate gyrus, where it was accompanied by the presence of degenerating neurons and loss of synaptic vesicle marker (synaptophysin). These data document that exposure to thimerosal during early postnatal life produces lasting alterations in the densities of brain opioid receptors along with other neuropathological changes, which may disturb brain development.     

Characterization of cytotoxic and genotoxic effects of different compounds in CHO K5 cells with the comet assay (single-cell gel electrophoresis assay)

Different variants of the comet assay were used to study the genotoxic and cytotoxic properties of the following eight compounds: chloral hydrate, colchicine, hydroquinone, DL-menthol, mitomycin C, sodium iodoacetate, thimerosal and valinomycin. Colchicine, mitomycin C, sodium iodoacetate and thimerosal induced genotoxic effects. The other compounds were found to be inactive. The compounds were tested in the standard comet assay as well as in the all cell comet assay (recovery of floating cells after treatment), designed in our laboratory for adherently-growing cells. This latter procedure proved to be more adequate for the assessment of the cytotoxicity for some of the compounds tested (hydroquinone, DL-menthol, thimerosal, valinomycin). Colchicine was positive in the standard comet assay (3h treatment) and in the all cell comet assay (24h treatment). Sodium iodoacetate and thimerosal were positive in the standard and/or the all cell comet assay. Chloral hydrate, hydroquinone, sodium iodoacetate, mitomycin C and thimerosal were also tested in the modified comet assay using lysed cells. Mitomycin C and thimerosal showed effects in this assay, whereas sodium iodoacetate was inactive. This indicates that it does not induce direct DNA damage. Compounds that are known or suspected to form DNA-DNA cross-links or DNA-protein cross-links (chloral hydrate, hydroquinone, mitomycin C and thimerosal) were checked for their ability to reduce ethyl methanesulfonate (EMS)-induced DNA damage. This mode of action could be demonstrated for mitomycin C only.     

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

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 μg/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 μg/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.     

Neurodevelopmental disorders after thimerosal-containing vaccines: a brief communication

We were initially highly skeptical that differences in the concentrations of thimerosal in vaccines would have any effect on the incidence rate of neurodevelopmental disorders after childhood immunization. This study presents the first epidemiologic evidence, based upon tens of millions of doses of vaccine administered in the United States, that associates increasing thimerosal from vaccines with neurodevelopmental disorders. Specifically, an analysis of the Vaccine Adverse Events Reporting System (VAERS) database showed statistical increases in the incidence rate of autism (relative risk [RR] = 6.0), mental retardation (RR = 6.1), and speech disorders (RR = 2.2) after thimerosal-containing diphtheria, tetanus, and acellular pertussis (DTaP) vaccines in comparison with thimerosal-free DTaP vaccines. The male/female ratio indicated that autism (17) and speech disorders (2.3) were reported more in males than females after thimerosal-containing DTaP vaccines, whereas mental retardation (1.2) was more evenly reported among male and female vaccine recipients. Controls were employed to determine if biases were present in the data, but none were found. It was determined that overall adverse reactions were reported in similar-aged populations after thimerosal-containing DTaP (2.4 +/- 3.2 years old) and thimerosal-free DTaP (2.1 +/- 2.8 years old) vaccinations. Acute control adverse reactions such as deaths (RR = 1.0), vasculitis (RR = 1.2), seizures (RR = 1.6), ED visits (RR = 1.4), total adverse reactions (RR = 1.4), and gastroenteritis (RR = 1.1) were reported similarly after thimerosal-containing and thimerosal-free DTaP vaccines. An association between neurodevelopmental disorders and thimerosal-containing DTaP vaccines was found, but additional studies should be conducted to confirm and extend this study.     

Thimerosal induces calcium mobilization, fructose 2,6-bisphosphate synthesis and cytoplasmic alkalinization in rat thymus lymphocytes

The effect of thimerosal on intracellular calcium ([Ca2+]i), pH (pHi) and fructose 2,6-bisphosphate (Fru 2,6-P2) in thymus lymphocytes was investigated. The effect of thimerosal on cell growth was also examined. Thimerosal produced a dose-dependent increase in [Ca2+]i, pHi and in the level of fructose 2,6-bisphosphate. Thimerosal was, however, unable to produce cell proliferation and inhibited [3H]thymidine incorporation when cells were challenged with PHA and costimulator. In the absence of external calcium, thimerosal produced only a slight increase in [Ca2+]i. In Na(+)-containing buffer, thimerosal induced an initial acidification (0.05 +/- 0.01 pH units), followed by an alkalinization of 0.08 pH units/min, whereas in Na(+)-free media, pHi decreased 0.2 +/- 0.02 units and this acidification was maintained for more than 40 min. When external calcium was removed the initial acidification was unchanged and no further increase in pHi was observed. Polymyxin B, an inhibitor of protein kinase C, did not modify the initial thimerosal-induced acidification although pH returned to basal levels after 10 min. It was concluded that alkalinization induced by thimerosal is probably due to activation of the Na+/H+ exchanger and that changes in internal Ca2+, pH and metabolic rate are not sufficient to induce cellular proliferation. The mechanism by which thimerosal inhibits thymocyte proliferation remains to be clarified.     

Evidence for thimerosal induced cytoprotection on rat stomach

Intragastrically administered thimerosal protects against ethanol induced ulceration with an ID50 of 1.6 mg/kg body weight. In contrast, intravenously administered thimerosal exhibits an ID50 of greater than 30 mg/kg. The antiulcerogenic effect of thimerosal persists for at least 20 hours before allowing gastric ulcers to be induced by ethanol. Intragastric application of thimerosal protects against acetyl salicylic acid induced with an ID50 of 6.8 mg/kg. Stress ulceration is inhibited by thimerosal with an ID50 of 9.2 mg/kg body weight. Investigations on the structure-activity relationship show that it is the mercury moiety and not the thiosalicylic acid moiety which is responsible for the inhibition by thimerosal.     

Thimerosal-Induced Apoptosis in Mouse C2C12 Myoblast Cells Occurs through Suppression of the PI3K/Akt/Survivin Pathway

Background

Thimerosal, a mercury-containing preservative, is one of the most widely used preservatives and found in a variety of biological products. Concerns over its possible toxicity have reemerged recently due to its use in vaccines. Thimerosal has also been reported to be markedly cytotoxic to neural tissue. However, little is known regarding thimerosal-induced toxicity in muscle tissue. Therefore, we investigated the cytotoxic effect of thimerosal and its possible mechanisms on mouse C2C12 myoblast cells.

Methodology/Principal Findings

The study showed that C2C12 myoblast cells underwent inhibition of proliferation and apoptosis after exposure to thimerosal (125–500 nM) for 24, 48 and 72 h. Thimerosal caused S phase arrest and induced apoptosis as assessed by flow cytometric analysis, Hoechst staining and immunoblotting. The data revealed that thimerosal could trigger the leakage of cytochrome c from mitochondria, followed by cleavage of caspase-9 and caspase-3, and that an inhibitor of caspase could suppress thimerosal-induced apoptosis. Thimerosal inhibited the phosphorylation of Akt^ser473 and survivin expression. Wortmannin, a PI3K inhibitor, inhibited Akt activity and decreased survivin expression, resulting in increased thimerosal-induced apoptosis in C2C12 cells, while the activation of PI3K/Akt pathway by mIGF-I (50 ng/ml) increased the expression of survivin and attenuated apoptosis. Furthermore, the inhibition of survivin expression by siRNA enhanced thimerosal-induced cell apoptosis, while overexpression of survivin prevented thimerosal-induced apoptosis. Taken together, the data show that the PI3K/Akt/survivin pathway plays an important role in the thimerosal-induced apoptosis in C2C12 cells.

Conclusions/Significance

Our results suggest that in C2C12 myoblast cells, thimerosal induces S phase arrest and finally causes apoptosis via inhibition of PI3K/Akt/survivin signaling followed by activation of the mitochondrial apoptotic pathway.     

Effect of preservatives on IgG aggregation, complement-activating effect and hypotensive activity of horse polyvalent antivenom used in snakebite envenomation.

Intravenous administration of antivenoms is associated with early adverse reactions in a number of cases, but the causes of this phenomenon are still unclear. The effect of preservatives (phenol and thimerosal) on IgG aggregate and dimer formation, in vitro complement-activating effect and hypotensive activity of a whole IgG horse liquid polyvalent antivenom, produced by caprylic acid fractionation, was assessed. These parameters were studied since they have been associated with the development of early adverse reactions to the administration of antivenoms and human immunoglobulins. After a three-year storage period at 4 degrees C, antivenoms with preservatives had an increased content of IgG aggregates and dimers when compared with antivenom devoid of phenol and thimerosal. These observations correlate with a slight increment in the turbidity of preservative-containing antivenoms. The three antivenoms studied (formulation: no preservatives; with phenol and thimerosal; with thimerosal alone) activated human complement in vitro, with only minor quantitative differences among them. When antivenoms were administered as a bolus intravenous injection in rats, a rapid and prominent hypotension of short duration was observed after injection of phenol-containing antivenom, whereas such an effect was absent in antivenom free of preservative and in the one containing only thimerosal. Bolus injection of saline solution with phenol resulted in a similar hypotension, indicating that the effect is due to phenol. However, when phenol-containing antivenom was diluted 1:5 with saline solution before infusion, as occurs in the clinical use of this product, no hypotension was observed. Our results stress the need to evaluate the effects of preservatives on the physicochemical and pharmacological characteristics of antivenoms.     

The thiol reagent, thimerosal, evokes Ca2+ spikes in HeLa cells by sensitizing the inositol 1,4,5-trisphosphate receptor.

The thiol reagent, thimerosal, has been shown to cause an increase in intracellular Ca2+ concentration ([Ca2+]i) in several cell types, and to cause Ca2+ spikes in unfertilized hamster eggs. Using single cell video-imaging we have shown that thimerosal evokes repetitive Ca2+ spikes in intact Fura-2-loaded HeLa cells that were similar in shape to those stimulated by histamine. Both thimerosal- and histamine-stimulated Ca2+ spikes occurred in the absence of extracellular (Ca2+ o), suggesting that they result from mobilization of Ca2+ from intracellular stores. Whereas histamine stimulated formation of inositol phosphates, thimerosal, at concentrations that caused sustained Ca2+ spiking, inhibited basal and histamine-stimulated formation of inositol phosphates. Thimerosal-evoked Ca2+ spikes are therefore not due to the stimulated production of inositol 1,4,5-trisphosphate (InsP3). The effects of thimerosal on Ca2+ spiking were probably due to alkylation of thiol groups on intracellular proteins because the spiking was reversed by the thiol-reducing compound dithiothreitol, and the latency between addition of thimerosal and a rise in [Ca2+]i was greatly shortened in cells where the intracellular reduced glutathione concentration had been decreased by preincubation with DL-buthionine (S,R)-sulfoximine. In permeabilized cells, thimerosal caused a concentration-dependent inhibition of Ca2+ accumulation, which was entirely due to inhibition of Ca2+ uptake into stores because thimerosal did not affect unidirectional 45Ca2+ efflux from stores preloaded with 45Ca2+. Thimerosal also caused a concentration-dependent sensitization of InsP3-induced Ca2+ mobilization: half-maximal mobilization of Ca2+ stores occurred with 161 +/- 20 nM InsP3 in control cells and with 62 +/- 5 nM InsP3 after treatment with 10 microM thimerosal. We conclude that thimerosal can mimic the effects of histamine on intracellular Ca2+ spiking without stimulating the formation of InsP3 and, in light of our results with permeabilized cells, suggest that thimerosal stimulates spiking by sensitizing cells to basal InsP3 levels.     

Cytogenetic control over the safety of merthiolate

The capacity of merthiolate (also known as thimerosal), a mercury organic compound used as preservative in vaccines, to induce chromosomal aberrations was studied in vivo in experiments on mice. The metaphasic method of the registration of anomalies was used. In a dose of 3.3 micrograms/kg body weight merthiolate did not produce a damaging effect on the chromosomes of marrow, spleen and embryonal liver cells. Cyclophosphamide (in a dose of 25-50 micrograms/kg) used as positive control induced a significant rise in the frequency of chromosomal transformations.     

Thimerosal increases the responsiveness of the calcium receptor in human parathyroid and rMTC6-23 cells

Parathyroid cells express a plasma membrane calcium receptor (CaR), which is stimulated by a rise in extracellular calcium concentration ([Ca2+]ext). A decreased sensitivity to [Ca2+]ext occurs in adenomatous parathyroid cells in patients with primary hyperparathyroidism, but the underlying functional mechanism is not yet fully understood. This study explored whether CaR responsiveness is influenced by increasing the affinity of IP3 receptors--a major signalling component of other G-protein-coupled receptors. The sulphydryl reagent thimerosal was used to increase the responsiveness of IP3-receptors. Quantitative fluorescence microscopy in Fura-2-loaded cells was used to investigate the effects of thimerosal on the cytoplasmic calcium concentrations ([Ca2+]i) in human parathyroid cells and to compare its effects in a rat medullary thyroid carcinoma cell line (rMTC6-23) also expressing CaR. During incubation in Ca(2+)-free medium, thimerosal 5 microM induced a rapid sustained rise in [Ca2+]i in human parathyroid cells and no further [Ca2+]i increase appeared in response to the CaR agonist Gd3+ (100 microM). Thimerosal 1 microM induced only slow and minimal changes of basal [Ca2+]i and allowed a rapid response to Gd3+ 20 nM (a concentration without effect in control cells). The slope of the thimerosal-induced [Ca2+]i responses was steeper following exposure to CaR agonists. In the presence of 1 mM [Ca2+]ext, thimerosal (0.5 microM) induced a sharp increase in [Ca2+]i to a peak (within 60 s), followed either by return to basal [Ca2+]i or by a plateau of slightly higher amplitude. Similar results were obtained using rMTC6-23 cells. Thimerosal increases the responsiveness to CaR agonists through modulation of the sensitivity of the IP3 receptor in both parathyroid and rMTC6-23 cells.     

Progress in Brucella vaccine development

Brucella spp. are zoonotic, facultative intracellular pathogens, which cause animal and human disease. Animal disease results in abortion of fetuses; in humans, it manifests flu-like symptoms with an undulant fever, with osteoarthritis as a common complication of infection. Antibiotic regimens for human brucellosis patients may last several months and are not always completely effective. While there are no vaccines for humans, several licensed live Brucella vaccines are available for use in livestock. The performance of these animal vaccines is dependent upon the host species, dose, and route of immunization. Newly engineered live vaccines, lacking well-defined virulence factors, retain low residual virulence, are highly protective, and may someday replace currently used animal vaccines. These also have possible human applications. Moreover, due to their enhanced safety and efficacy in animal models, subunit vaccines for brucellosis show great promise for their application in livestock and humans. This review summarizes the progress of brucellosis vaccine development and presents an overview of candidate vaccines.