Bei Thiomersal handelt es sich um ein quecksilberhaltiges Konservierungsmittel in Medizinprodukten, welches auch in Impfstoffen Verwendung findet. In dieser Arbeit wurden die Toxizität von Thiomersal und von Glutathionkonjugaten seiner Dissoziationsprodukte sowie zum Vergleich die Toxizität von zwei strukturell mit Thiomersal bzw. seinen Metaboliten verwandten Referenzsubstanzen, p-Chlormercuribenzoesäure und Ethylquecksilberchlorid, in Zellsystemen mit humanen Zellen vom Typ A-549 und Hep-G2 untersucht. Alle fünf Substanzen führten zu einer dosisabhängigen Vitalitätsreduktion bei beiden Zelllinien. Die stärkste Vitalitätsreduktion wurde durch Ethylquecksilberchlorid erzielt, danach folgten in absteigender Reihenfolge Thiomersal, Ethylquecksilber-L-Glutathion und p-Chlormercuribenzoesäure. Den schwächsten vitalitätsreduzierenden Effekt zeigte Thiosalicyl-L-Glutathion-Disulfid. Diese Ergebnisse sprechen dafür, dass die Glutathion-Konjugation von Thiomersal zu einer Abschwächung der Toxizität im menschlichen Organismus führt und untermauern die Annahme einer protektiven Wirkung von Glutathion. Die giftende Rolle von Glutathion in Bioaktivierungsprozessen anderer Noxen wird diskutiert. Es wurde im Rahmen dieser Arbeit ein Beitrag zum besseren Verständnis der Toxizität von Thiomersal im humanen Organismus geleistet und darüber hinaus Bezüge zum potentiellen allergenen Potential von Thiomersal hergestellt.

Bei Thiomersal handelt es sich um ein quecksilberhaltiges Konservierungsmittel in Medizinprodukten, welches auch in Impfstoffen Verwendung findet. In dieser Arbeit wurden die Toxizität von Thiomersal und von Glutathionkonjugaten seiner Dissoziationsprodukte sowie zum Vergleich die Toxizität von zwei strukturell mit Thiomersal bzw. seinen Metaboliten verwandten Referenzsubstanzen, p-Chlormercuribenzoesäure und Ethylquecksilberchlorid, in Zellsystemen mit humanen Zellen vom Typ A-549 und Hep-G2 untersucht. Alle fünf Substanzen führten zu einer dosisabhängigen Vitalitätsreduktion bei beiden Zelllinien. Die stärkste Vitalitätsreduktion wurde durch Ethylquecksilberchlorid erzielt, danach folgten in absteigender Reihenfolge Thiomersal, Ethylquecksilber-L-Glutathion und p-Chlormercuribenzoesäure. Den schwächsten vitalitätsreduzierenden Effekt zeigte Thiosalicyl-L-Glutathion-Disulfid. Diese Ergebnisse sprechen dafür, dass die Glutathion-Konjugation von Thiomersal zu einer Abschwächung der Toxizität im menschlichen Organismus führt und untermauern die Annahme einer protektiven Wirkung von Glutathion. Die giftende Rolle von Glutathion in Bioaktivierungsprozessen anderer Noxen wird diskutiert. Es wurde im Rahmen dieser Arbeit ein Beitrag zum besseren Verständnis der Toxizität von Thiomersal im humanen Organismus geleistet und darüber hinaus Bezüge zum potentiellen allergenen Potential von Thiomersal hergestellt.

Bei Thiomersal handelt es sich um ein quecksilberhaltiges Konservierungsmittel in Medizinprodukten, welches auch in Impfstoffen Verwendung findet. In dieser Arbeit wurden die Toxizität von Thiomersal und von Glutathionkonjugaten seiner Dissoziationsprodukte sowie zum Vergleich die Toxizität von zwei strukturell mit Thiomersal bzw. seinen Metaboliten verwandten Referenzsubstanzen, p-Chlormercuribenzoesäure und Ethylquecksilberchlorid, in Zellsystemen mit humanen Zellen vom Typ A-549 und Hep-G2 untersucht. Alle fünf Substanzen führten zu einer dosisabhängigen Vitalitätsreduktion bei beiden Zelllinien. Die stärkste Vitalitätsreduktion wurde durch Ethylquecksilberchlorid erzielt, danach folgten in absteigender Reihenfolge Thiomersal, Ethylquecksilber-L-Glutathion und p-Chlormercuribenzoesäure. Den schwächsten vitalitätsreduzierenden Effekt zeigte Thiosalicyl-L-Glutathion-Disulfid. Diese Ergebnisse sprechen dafür, dass die Glutathion-Konjugation von Thiomersal zu einer Abschwächung der Toxizität im menschlichen Organismus führt und untermauern die Annahme einer protektiven Wirkung von Glutathion. Die giftende Rolle von Glutathion in Bioaktivierungsprozessen anderer Noxen wird diskutiert. Es wurde im Rahmen dieser Arbeit ein Beitrag zum besseren Verständnis der Toxizität von Thiomersal im humanen Organismus geleistet und darüber hinaus Bezüge zum potentiellen allergenen Potential von Thiomersal hergestellt.

The effects of 1 min 4 hr exposures to mercuric chloride (HgCl2), methyl mercuric chloride (CH3HgCl), p-chloromercuribenzoate (p-CMB) and ethylmercurithiosalicylate (TMS) on cell viability and kinetics of cell death, microtubules, F-actin, CD3 receptor expression, protein tyrosine phosphorylation (PTyr-P), intracellular calcium [Ca2+]i and responses to polarized signals in YAC-1 lymphoma cells were investigated. We hypothesized that immunotoxic effects of HgCl2 (Hg2+) are initiated by global receptor triggering, accompanied by increased protein tyrosine phosphorylation (PTyr-P) and down-regulation of the T-cell receptor (TCR). As a polychloride anion with poor lipid solubility, inorganic Hg2+ may produce effects at the outer cell membrane before significant intracellular accumulation, loss of microtubule integrity (a sensitive target) and activation of cell death through apoptotic pathways. The organomercurial compound p-CMB is likewise thought to penetrate membranes slowly as a result of ionization. In contrast, the highly lipid-soluble organomercurial compounds CH3HgCl and TMS were expected to reduce responses to polarized stimuli only in conjunction with and not prior to loss of microtubule integrity and the onset of necrotic cell death. <p>Two general patterns of effects were observed. In HgCl2-treated YAC-1 cells, inhibition of responses to polarized stimuli preceded loss of microtubules and onset of cell death. Effects on polarized stimuli were preceded by a transient Ca2+ signal; however, this Ca2+ signal appeared abortive, accompanied by a paradoxic decrease in PTyr-P and partial down-regulation of CD3 receptors. Responses to polarised stimuli were inhibited prior to extensive loss of microtubule staining, indicating effects preceded cytosolic Hg2+ accumulation. HgCl2 exposure was followed rapidly by necrotic cell death. <p>Similarly, p-CMB-treated YAC-1 cells failed to respond to polarized stimuli before effects on microtubules or loss of viability, and proceeded rapidly to late apoptosis; however, a transient Ca2+ signal and progressive loss of F-actin preceded effects in all other assays and may account for loss of polarized responses. <p>In CH3HgCl- and TMS-treated YAC-1 cells, CD3 receptor expression, [Ca2+] and PTyr-P were increased immediately, along with loss of microtubules. These reductions preceded inhibition of polarized signaling responses and seemed to indicate a general loss of cellular homeostasis not seen in HgCl2- and p-CMB-treated cells; loss of homeostasis did not necessarily produce simultaneous loss of viability, as TMS-treated cells remained viable for 30 min while CH3HgCl-treated cells became apoptotic within 1 min. Nonetheless, the YAC-1 cells proceeded to cell death more slowly, remaining early apoptotic after 4 hr, when almost all HgCl2- and p-CMB-treated cells were necrotic. These findings indicate the two groups of mercury compounds may alter responses to polarized stimuli and induce cell death by distinct pathways, one involving an apparently abortive signal and the other mediated by much more profound disruption of cellular homeostasis. Within the larger patterns there are further differences between the effects produced by each Hg compound, likely reflecting the combined influence of pharmacokinetic and dynamic factors governing access to and interactions with different cellular targets leading to cell death. These distinct targets may in turn be reflected in the different immune effects produced by these compounds <i>in vivo</i>.

The effects of 1 min 4 hr exposures to mercuric chloride (HgCl2), methyl mercuric chloride (CH3HgCl), p-chloromercuribenzoate (p-CMB) and ethylmercurithiosalicylate (TMS) on cell viability and kinetics of cell death, microtubules, F-actin, CD3 receptor expression, protein tyrosine phosphorylation (PTyr-P), intracellular calcium [Ca2+]i and responses to polarized signals in YAC-1 lymphoma cells were investigated. We hypothesized that immunotoxic effects of HgCl2 (Hg2+) are initiated by global receptor triggering, accompanied by increased protein tyrosine phosphorylation (PTyr-P) and down-regulation of the T-cell receptor (TCR). As a polychloride anion with poor lipid solubility, inorganic Hg2+ may produce effects at the outer cell membrane before significant intracellular accumulation, loss of microtubule integrity (a sensitive target) and activation of cell death through apoptotic pathways. The organomercurial compound p-CMB is likewise thought to penetrate membranes slowly as a result of ionization. In contrast, the highly lipid-soluble organomercurial compounds CH3HgCl and TMS were expected to reduce responses to polarized stimuli only in conjunction with and not prior to loss of microtubule integrity and the onset of necrotic cell death. <p>Two general patterns of effects were observed. In HgCl2-treated YAC-1 cells, inhibition of responses to polarized stimuli preceded loss of microtubules and onset of cell death. Effects on polarized stimuli were preceded by a transient Ca2+ signal; however, this Ca2+ signal appeared abortive, accompanied by a paradoxic decrease in PTyr-P and partial down-regulation of CD3 receptors. Responses to polarised stimuli were inhibited prior to extensive loss of microtubule staining, indicating effects preceded cytosolic Hg2+ accumulation. HgCl2 exposure was followed rapidly by necrotic cell death. <p>Similarly, p-CMB-treated YAC-1 cells failed to respond to polarized stimuli before effects on microtubules or loss of viability, and proceeded rapidly to late apoptosis; however, a transient Ca2+ signal and progressive loss of F-actin preceded effects in all other assays and may account for loss of polarized responses. <p>In CH3HgCl- and TMS-treated YAC-1 cells, CD3 receptor expression, [Ca2+] and PTyr-P were increased immediately, along with loss of microtubules. These reductions preceded inhibition of polarized signaling responses and seemed to indicate a general loss of cellular homeostasis not seen in HgCl2- and p-CMB-treated cells; loss of homeostasis did not necessarily produce simultaneous loss of viability, as TMS-treated cells remained viable for 30 min while CH3HgCl-treated cells became apoptotic within 1 min. Nonetheless, the YAC-1 cells proceeded to cell death more slowly, remaining early apoptotic after 4 hr, when almost all HgCl2- and p-CMB-treated cells were necrotic. These findings indicate the two groups of mercury compounds may alter responses to polarized stimuli and induce cell death by distinct pathways, one involving an apparently abortive signal and the other mediated by much more profound disruption of cellular homeostasis. Within the larger patterns there are further differences between the effects produced by each Hg compound, likely reflecting the combined influence of pharmacokinetic and dynamic factors governing access to and interactions with different cellular targets leading to cell death. These distinct targets may in turn be reflected in the different immune effects produced by these compounds <i>in vivo</i>.

Interaction between bovine serum albumin (BSA) and thimerosal (TM), organic mercury compound, was investigated by spectroscopic methods. The results, by molecular fluorescence, show that the interaction takes place by static quenching with electrostatic forces spontaneously (ΔG = - 4.40 kJ mol-1 at 30°C). The binding constant (Kb) was 3.24 ± 0.01x103 L mol-1 (30°C) is considered a moderate interaction. Fluorescence in three dimensions revealed that TM causes structural involving the the polypeptide chain BSA changes in the polarity of the tryptophan and tyrosine residues confirmed by circular dichroism (CD) showed an increase in α-helix content after interaction with TM. In addition, the TM decreases the surface hydrophobicity of the protein. Bilirubin was used as a marker for the subdomain IB, confirming that TM interacts in this region of the protein. The study of the interaction mechanism proposed that TM is reacted with BSA through the free cysteine residue, forming the adduct BSA-HgEt release of thiosalicylic acid (ATS), which interacts with amino acids with side chain positive. Besides, it was seen that TM accelerates the protein fibrillation kinetics by 42%, with a possible indication of the toxicity of this compound in biological systems. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A interação entre albumina do soro bovino (BSA) e timerosal (TM), composto orgânico de mercúrio, foi investigada utilizando métodos espectroscópicos. Os resultados, por fluorescência molecular, evidenciam que a interação acontece por quenching estático através de forças eletrostáticas de forma espontânea (ΔG = - 4,40 kJ mol-1 a 30ºC). A constante de ligação (Kb) foi de 3,24 ± 0,01x103 L mol-1 (30ºC) sendo considerada uma interação moderada. A fluorescência em três dimensões revelou que TM causa mudanças estruturais envolvendo a cadeia polipeptídica da BSA assim como altera a polaridade dos resíduos de triptofano e tirosina, confirmada por dicroísmo circular (DC) que evidenciou aumento no conteúdo de α-hélice após interação com TM. Além disto, TM diminui a hidrofobicidade superficial da proteína. Bilirrubina foi utilizada como marcador para o subdomínio IB, confirmando que TM interage nesta região da proteína. O estudo do mecanismo de interação propôs que TM reage com BSA através do resíduo de cisteína livre, formando o aduto BSA-HgEt com liberação de ácido tiosalicílico (ATS), que interage com os aminoácidos com cadeia lateral positiva. Por fim, foi visto que TM acelera a cinética de fibrilação proteica em 42%, sendo um possível indício da toxicidade deste composto em sistemas biológicos.

This thesis focuses on the implementation of species-specific isotope dilution (SSID) methodology and stable isotope tracers to determine mercury species occurrence and transformation processes in-situ and during sample treatment. Isotope enriched tracers of methyl-, ethyl- and inorganic mercury were synthesised and applied in different combinations to marine and biological samples. Experimental results were obtained using gas chromatography-inductively coupled plasma-mass spectrometry (GC-ICP-MS). Mercury methylation and methylmercury demethylation processes in surface sediments were studied in the brackish Öre River estuary, Bothnian Bay. Uni- and multivariate data evaluation identified the organic material content and mercury methylation potential in the sediments as important factors controlling incipient methylmercury levels. Mercury species distribution in mice treated with the pharmaceutical preservative Thimerosal (ethylmercurithiosalicylate) was studied. The ethylmercury moiety of Thimerosal was observed to rapidly convert to inorganic mercury in the mice during the treatment period as well as during sample treatment, hence necessitating SSID methodology for accurate ethylmercury determinations in biological samples. To facilitate the introduction of SSID as a routine quantitative method in mercury speciation, a methylmercury isotopic certified reference material (ICRM) was produced. Prior to certification, the stability of the material was examined in conventional and isochronous stability studies spanning 12 months, which permitted uncertainty estimation of the methylmercury amount content for two years of shelf-life. Finally, a field-adapted SSID method for methylmercury determinations in natural water samples was developed. The proposed analytical protocol significantly simplified sample storage- and treatment procedures without sacrifices in analytical accuracy.