O timerosal é um agente antisséptico utilizado em vacinas como conservante. Devido a presença de etilmercúrio (EtHg) em sua composição (com aproximadamente 49% de mercúrio (Hg) em peso), uma preocupação existe em relação aos possíveis efeitos tóxicos em humanos. No entanto, pouco se sabe sobre o perfil cinético do EtHg em mamíferos. Neste sentido, este trabalho teve como objetivo avaliar a distribuição tecidual e meias-vidas do Hg, seu metabolismo no sangue (conversão a mercúrio inorgânico) e nefrotoxicidade após exposição ao timerosal, utilizando modelos in vivo e in vitro. Para isto, o trabalho foi dividido em 3 estudos: (I) camundongos machos Swiss foram expostos a 20 ?g de Hg sob a forma de timerosal via intramuscular. Sangue, cérebro, coração, rim e fígado foram coletados após 0,5; 1; 8; 16; 144; 720 e 1980 horas (h) da exposição (n=4) e analisados quanto às concentrações das espécies de Hg por HPLC-ICP-MS; (II) alíquotas (n=4) de sangue total, plasma e eritrócitos humanos foram expostas a timerosal ou EtHg (3 mg/l) durante 24 h e analisadas quanto às concentrações das espécies de Hg por HPLC-ICP-MS; (III) células HK2 foram expostas durante 24 h a timerosal (0 ?M a 2 ?M) e avaliadas quanto à viabilidade e proliferação celular, apoptose, expressão de proteínas Bax e TGF-?1, saúde mitocondrial e concentrações de fibronectina no meio. Verificou-se que o transporte de EtHg do músculo para os tecidos e a sua conversão em Hg inorgânico (Hgi) ocorrem rapidamente. Após 0,5 h da exposição ao timerosal, as concentrações mais altas de ambos EtHg e Hgi foram encontradas no rins (> 70% do Hg total no corpo do animal). O cérebro apresentou uma menor contribuição para a carga corporal de Hg (<1,0% do Hg total no corpo do animal). Após trinta dias da exposição ao timerosal, houve excreção considerável de Hg e o fígado apresentou a maior parte do Hg ainda restante no corpo dos animais. As meias-vidas foram estimadas (em dias) em 8,8; 10,7; 7,8; 7,7 e 45,2; para o sangue, cérebro, coração, fígado e rim, ii respectivamente. Sugere-se que a extensão da conversão de EtHg a Hgi é modulada em parte pela partição do EtHg no plasma e no sangue, uma vez que o EtHg é rapidamente convertido a Hgi nas células vermelhas, mas não no plasma. O mecanismo de dealquilação em células vermelhas parece ser mediado pela reação de Fenton (formação de radicais hidroxil). Ainda, EtHg/timerosal diminuiu a viabilidade celular e mitose, promoveu a apoptose, prejudicou o estado de transição de permeabilidade mitocondrial, aumentou a expressão de Bax e TGF-?1 e secreção de fibronectina. Coletivamente, os resultados demonstram que a cinética do timerosal (EtHg) está mais próxima a do Hgi - e não do MeHg - e o rim deve ser considerado um alvo potencial de toxicidade do EtHg, já que é o órgão exposto às maiores concentrações de Hg , e onde o metal apresenta sua maior meia-vida biológica. Adicionalmente, o EtHg/timerosal demonstrou ser um agente antiproliferativo, apoptótico e pró-fibrótico em células humanas renais. / Thimerosal is an antiseptic agent used in vaccines as a preservative. Due to the presence of ethylmercury (EtHg) in its composition (approximately 49% mercury (Hg) by weight), there is a concern regarding possible toxic effects in humans. However, little is known about the kinetic profile of EtHg in mammals. Thus, this study aimed to evaluate the tissue distribution and half-life of Hg, its metabolism in blood (conversion to inorganic mercury) and nephrotoxicity after exposure to thimerosal, using in vivo and in vitro models. The work was divided into three studies: (I) Swiss male mice were exposed to 20 ?g of Hg in the form of thimerosal intramuscularly. Blood, brain, heart, kidney and liver were collected after 0.5, 1, 8, 16, 144, 720 and 1980 hours (h) of exposure (n=4) and analyzed for concentrations of Hg species by HPLC-ICP-MS; (II) aliquots (n=4) of whole blood, plasma and erythrocytes were exposed to thimerosal or EtHg (3 mg/l) for 24 h and analyzed for Hg species concentrations by HPLC-ICP-MS; (III) HK2 cells were exposed for 24 h to thimerosal (0 ?M to 2 ?M) and evaluated for cell viability and proliferation, apoptosis, Bax and TGF-?1 expression, mitochondrial health and concentrations of fibronectin in the medium. It has been found that the transport of EtHg from muscle to tissues and its conversion into inorganic Hg (Hgi) occur quickly. After 0.5 h of exposure to thimerosal, higher concentrations of both EtHg and Hgi were found in the kidneys (> 70% of total Hg in the animal body). The brain showed a minor contribution to the body burden of Hg (<1.0% of total Hg in the animal body). Thirty days after exposure to thimerosal, there was considerable excretion of Hg and liver had the most Hg still remaining in the animal body. Half-lives were estimated (in days) at 8.8; 10.7; 7.8; 7.7 and 45.2; for blood, brain, heart, liver and kidney, respectively. It is suggested that the extent of conversion of the EtHg into Hgi is modulated in part by the partition of EtHg in plasma and blood, since EtHg is rapidly converted into Hgi in red cells but not in plasma. The mechanism of dealkylation in red cells seems to be mediated by the Fenton reaction iv (formation of hydroxyl radicals). Additionally, EtHg/thimerosal decreased cell viability and mitosis, promoted apoptosis, impaired mitochondrial permeability transition state, increased expression of Bax and TGF-?1 and fibronectin secretion in the media. Collectively, the results demonstrate that the kinetics of thimerosal (EtHg) is closer to the Hgi - and not the MeHg - and kidney should be considered a potential target for EtHg toxicity, since it is exposed to the highest concentrations of Hg and it is the tissue where the metal has a greater biological half-life. Additionally, EtHg/thimerosal was shown to be an antiproliferative, apoptotic and pro-fibrotic agent in human kidney cells.

The organic mercury compound ethylmercurithiosalicylate (thimerosal), an antiseptic and a preservative, has recently raised public health concern due to its presence in vaccines globally. Thimerosal dissociates in the body to thiosalicylate and ethyl mercury (EtHg), which is partly converted to inorganic mercuric mercury (Hg2+). The immunosuppressive, immunostimulatory, and de novo autoimmunogen effect of thimerosal in mice, as well as the accelerating/aggravating effect on spontaneous systemic autoimmunity including dose-response aspects were the subject of this thesis. Thimerosal perorally (590 μg Hg/kg body weight (bw)/day) to genetically susceptible (H-2s) mice caused immunosuppression during the first week with reduction of the total number of splenocytes, T- and B-cells. The suppression lasted 2 weeks for CD4+ cells, but was superseded by a strong immunostimulation/proliferation including T- as well as B-cells, and polyclonal B-cell activation (PBA). Antinuclear antibodies targeting the 34-kDa nucleolar protein fibrillarin (AFA) appeared after 10 days, followed by renal mesangial and systemic vessel wall immune-complex (IC) deposits. The Lowest Observed Adverse Effect Level (LOAEL) was in the order AFA = glomerular and splenic vessel wall deposits &lt; hyperimmunoglobulinemia &lt; PBA. The LOAEL for AFA was 118 μg Hg/kg bw/day. The LOAEL for the different parameters of this thimerosal-induced systemic autoimmune condition (HgIA) was 3-11-fold higher compared with HgIA induced by HgCl2. The thimerosal-induced HgIA shared with HgCl2 a significant dose-response relationship, and requirement for: T-cells, the costimulatory factor CD28, the IFN-γ/IFN-γ-receptor pathway,but not IL-4. The mRNA expression in lymph nodes of IL-2, IFN-γ, IL-4, and IL-15 was significantly increased but not delayed compared with HgCl2. Treatment with the ubiquitous organic Hg compound methyl Hg using equimolar doses of Hg (533 μg Hg/kg bw/day) caused a transient immunosuppression, followed by a weak immunostimulation and AFA. The IgG AFA isotypes induced by the organic Hg compounds MeHg and EtHg were stable and dominated by a Th1-like pattern over a broad time- and dose range. Treatment with inorganic HgCl2 caused a dose- and time-dependent pattern of IgG AFA isotypes. Low doses favored a Th1-like pattern, a high dose a balanced or Th2-like pattern. Middle-range doses showed initially a Th1-like pattern which gradually evolved into a balanced or Th2-like pattern. The qualitative difference in IgG AFA isotypes between organic and inorganic Hg may be due to differences in activation and/or suppression of T-helper cell subsets or factors influencing the Th1/Th2-function. Speciation of the renal Hg2+ concentration and comparison with the threshold dose for induction of AFA by HgCl2 showed that even with the lowest doses of thimerosal and MeHg used in this thesis, the AFA response might from a dose threshold point of view have been caused by conversion of the organic Hg species to Hg2+. Primary treatment with inorganic Hg (HgCl2) accelerates/aggravates murine systemic autoimmunity, both spontaneous (genetic) and induced by other means. This capacity was assessed for thimerosal over a broad dose range using the (NZB X NZW)F1 hybrid mouse model. Significantly increased antinuclear antibodies (ANA) was seen after 4-7 weeks treatment (LOAEL 147 μg Hg/kg bw/day), and the response was dose-dependent up to 13 weeks. Renal mesangial and systemic vessel walls deposits similar to those in de novo HgIA were present after 7 weeks treatment. Twenty-two to 25 weeks treatment with thimerosal caused, in a dose-dependent fashion (LOAEL 295 μg Hg/kg bw/day), relocalization of the spontaneously developing glomerular IC deposits from the capillary vessel walls to the mesangium, which attenuated histological kidney damage and proteinuria, and increased survival. Thimerosal caused systemic vessel wall IC-deposits over a broad dose range: the Low Observed Adverse Effect Level (LOAEL) for renal and splenic vessel wall IC deposits was 18 and 9 μg Hg/kg bw/day, respectively. The No Observed Adverse Effect Level (NOAEL) could not be determined for the latter, since deposits were present even with the lowest dose used. Thimerosal causes in genetically susceptible mice an initial, transient immunosuppression which is superseded by a strong immunostimulation and systemic autoimmunity, sharing many characteristics with the HgIA induced by inorganic HgCl2. The IgG AFA isotype pattern is however qualitatively different, and the threshold dose substantially higher. In contrast, long-term treatment with thimerosal induces systemic vessel wall IC-deposits also using doses below those needed to induce HgIA de novo in H-2s mice.

O timerosal é um agente antisséptico utilizado em vacinas como conservante. Devido a presença de etilmercúrio (EtHg) em sua composição (com aproximadamente 49% de mercúrio (Hg) em peso), uma preocupação existe em relação aos possíveis efeitos tóxicos em humanos. No entanto, pouco se sabe sobre o perfil cinético do EtHg em mamíferos. Neste sentido, este trabalho teve como objetivo avaliar a distribuição tecidual e meias-vidas do Hg, seu metabolismo no sangue (conversão a mercúrio inorgânico) e nefrotoxicidade após exposição ao timerosal, utilizando modelos in vivo e in vitro. Para isto, o trabalho foi dividido em 3 estudos: (I) camundongos machos Swiss foram expostos a 20 ?g de Hg sob a forma de timerosal via intramuscular. Sangue, cérebro, coração, rim e fígado foram coletados após 0,5; 1; 8; 16; 144; 720 e 1980 horas (h) da exposição (n=4) e analisados quanto às concentrações das espécies de Hg por HPLC-ICP-MS; (II) alíquotas (n=4) de sangue total, plasma e eritrócitos humanos foram expostas a timerosal ou EtHg (3 mg/l) durante 24 h e analisadas quanto às concentrações das espécies de Hg por HPLC-ICP-MS; (III) células HK2 foram expostas durante 24 h a timerosal (0 ?M a 2 ?M) e avaliadas quanto à viabilidade e proliferação celular, apoptose, expressão de proteínas Bax e TGF-?1, saúde mitocondrial e concentrações de fibronectina no meio. Verificou-se que o transporte de EtHg do músculo para os tecidos e a sua conversão em Hg inorgânico (Hgi) ocorrem rapidamente. Após 0,5 h da exposição ao timerosal, as concentrações mais altas de ambos EtHg e Hgi foram encontradas no rins (> 70% do Hg total no corpo do animal). O cérebro apresentou uma menor contribuição para a carga corporal de Hg (<1,0% do Hg total no corpo do animal). Após trinta dias da exposição ao timerosal, houve excreção considerável de Hg e o fígado apresentou a maior parte do Hg ainda restante no corpo dos animais. As meias-vidas foram estimadas (em dias) em 8,8; 10,7; 7,8; 7,7 e 45,2; para o sangue, cérebro, coração, fígado e rim, ii respectivamente. Sugere-se que a extensão da conversão de EtHg a Hgi é modulada em parte pela partição do EtHg no plasma e no sangue, uma vez que o EtHg é rapidamente convertido a Hgi nas células vermelhas, mas não no plasma. O mecanismo de dealquilação em células vermelhas parece ser mediado pela reação de Fenton (formação de radicais hidroxil). Ainda, EtHg/timerosal diminuiu a viabilidade celular e mitose, promoveu a apoptose, prejudicou o estado de transição de permeabilidade mitocondrial, aumentou a expressão de Bax e TGF-?1 e secreção de fibronectina. Coletivamente, os resultados demonstram que a cinética do timerosal (EtHg) está mais próxima a do Hgi - e não do MeHg - e o rim deve ser considerado um alvo potencial de toxicidade do EtHg, já que é o órgão exposto às maiores concentrações de Hg , e onde o metal apresenta sua maior meia-vida biológica. Adicionalmente, o EtHg/timerosal demonstrou ser um agente antiproliferativo, apoptótico e pró-fibrótico em células humanas renais. / Thimerosal is an antiseptic agent used in vaccines as a preservative. Due to the presence of ethylmercury (EtHg) in its composition (approximately 49% mercury (Hg) by weight), there is a concern regarding possible toxic effects in humans. However, little is known about the kinetic profile of EtHg in mammals. Thus, this study aimed to evaluate the tissue distribution and half-life of Hg, its metabolism in blood (conversion to inorganic mercury) and nephrotoxicity after exposure to thimerosal, using in vivo and in vitro models. The work was divided into three studies: (I) Swiss male mice were exposed to 20 ?g of Hg in the form of thimerosal intramuscularly. Blood, brain, heart, kidney and liver were collected after 0.5, 1, 8, 16, 144, 720 and 1980 hours (h) of exposure (n=4) and analyzed for concentrations of Hg species by HPLC-ICP-MS; (II) aliquots (n=4) of whole blood, plasma and erythrocytes were exposed to thimerosal or EtHg (3 mg/l) for 24 h and analyzed for Hg species concentrations by HPLC-ICP-MS; (III) HK2 cells were exposed for 24 h to thimerosal (0 ?M to 2 ?M) and evaluated for cell viability and proliferation, apoptosis, Bax and TGF-?1 expression, mitochondrial health and concentrations of fibronectin in the medium. It has been found that the transport of EtHg from muscle to tissues and its conversion into inorganic Hg (Hgi) occur quickly. After 0.5 h of exposure to thimerosal, higher concentrations of both EtHg and Hgi were found in the kidneys (> 70% of total Hg in the animal body). The brain showed a minor contribution to the body burden of Hg (<1.0% of total Hg in the animal body). Thirty days after exposure to thimerosal, there was considerable excretion of Hg and liver had the most Hg still remaining in the animal body. Half-lives were estimated (in days) at 8.8; 10.7; 7.8; 7.7 and 45.2; for blood, brain, heart, liver and kidney, respectively. It is suggested that the extent of conversion of the EtHg into Hgi is modulated in part by the partition of EtHg in plasma and blood, since EtHg is rapidly converted into Hgi in red cells but not in plasma. The mechanism of dealkylation in red cells seems to be mediated by the Fenton reaction iv (formation of hydroxyl radicals). Additionally, EtHg/thimerosal decreased cell viability and mitosis, promoted apoptosis, impaired mitochondrial permeability transition state, increased expression of Bax and TGF-?1 and fibronectin secretion in the media. Collectively, the results demonstrate that the kinetics of thimerosal (EtHg) is closer to the Hgi - and not the MeHg - and kidney should be considered a potential target for EtHg toxicity, since it is exposed to the highest concentrations of Hg and it is the tissue where the metal has a greater biological half-life. Additionally, EtHg/thimerosal was shown to be an antiproliferative, apoptotic and pro-fibrotic agent in human kidney cells.

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[pt] O tiomersal é um composto organomercurial usado como conservante em vacinas. Sua degradação sob exposição luz visível ou ao UV produz ácido tiosalicílico, ácido ditiobenzóico e etilmercúrio. A inclusão do tiomersal em composições vacinais é controversa, pois alguns estudos apontam sua correlação com neurotoxicidade, porém, outras investigações ressaltam a sua segurança. O objetivo desse estudo foi avaliar o grau de degradação do tiomersal sob influência de luz, pH, temperatura e material da embalagem, mediante a comparação de três técnicas analíticas. Adicionalmente, foi avaliado o efeito de excipientes vacinais. Conduziu-se uma investigação, inicialmente por espectrofotometria de absorção molecular no UV-vis, em relação à foto-exposição e presença dos excipientes. Para apoiar a confiabilidade dos resultados, testes estatísticos foram realizados. Para a análise da formação de quantidades ultra-traço de espécies mercuriais (etilmercúrio e mercúrio inorgânico), técnicas de maior sensibilidade como a espectrometria de absorção atômica e de fluorescência atômica acoplada à cromatografia gasosa foram utilizadas. Os resultados revelaram degradação do tiomersal sob exposição ao UV. A presença de excipientes durante a foto-irradiação demonstrou efeitos variados na estabilidade do tiomersal, com alguns excipientes favorecendo a degradação. Além disso, o material do recipiente foi relevante e as variações de temperatura e pH não afetaram significativamente a estabilidade. O tiomersal manteve-se estável na maioria dos testes que envolvia condições reais de armazenamento de vacinas. Essas descobertas contribuem para a compreensão do comportamento do conservante frente a fatores potenciais que influenciam sua estabilidade, subsidiando o desenvolvimento e armazenamento de vacinas, garantindo sua eficácia e segurança. / [en] Thiomersal is an organomercury compound used as a preservative in vaccines. Its degradation under exposure to visible light or UV produces thiosalicylic acid, dithiobenzoic acid and ethylmercury. The inclusion of thiomersal in vaccine compositions is controversial, as some studies indicate its correlation with neurotoxicity, , however, other investigations emphasize its safety. The objective of this study was to evaluate the degree of degradation of thiomersal under the influence of light, pH, temperature and packaging material, by comparing three analytical techniques. Additionally, the effect of vaccine excipients was evaluated. An investigation was conducted, initially by UV-vis molecular absorption spectrophotometry, regarding photo-exposure and the presence of excipients. To support the reliability of the results, statistical tests were performed. To analyze the formation of ultra-trace amounts of mercury species (ethylmercury and inorganic mercury), highly sensitive techniques such as atomic absorption and atomic fluorescence spectrometry coupled to gas chromatography were used. The results revealed degradation of thiomersal under UV exposure. The presence of excipients during photoirradiation demonstrated varying effects on the stability of thiomersal, with some excipients favoring degradation. Furthermore, the container material was relevant and variations in temperature and pH did not significantly affect stability. Therefore, thiomersal remained stable in most tests involving real vaccine storage conditions. These findings contribute to the understanding of preservative behavior in the face of potential factors that influence its stability, supporting the development and storage of vaccines, ensuring their efficacy and safety.

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.