no / Nonsegmental vitiligo (NSV) is characterized by loss of inherited skin color. The cause of the disease is still unknown despite accumulating in vivo and in vitro evidence of massive epidermal oxidative stress via H2O2 and peroxynitrite (ONOO−) in affected individuals. The most favored hypothesis is based on autoimmune mechanisms. Strictly segmental vitiligo (SSV) with dermatomal distribution is a rare entity, often associated with stable outcome. Recently, it was documented that this form can be associated with NSV (mixed vitiligo). We here asked the question whether ROS and possibly ONOO− could be players in the pathogenesis of SSV. Our in situ results demonstrate for the first time epidermal biopterin accumulation together with significantly decreased epidermal catalase, thioredoxin/thioreoxin reductase, and MSRA/MSRB expression. Moreover, we show epidermal ONOO− accumulation. In vivo FT-Raman spectroscopy reveals the presence of H2O2, methionine sulfoxide, and tryptophan metabolites; i.e., N-formylkynurenine and kynurenine, implying Fenton chemistry in the cascade (n=10). Validation of the basic data stems from successful repigmentation of skin and eyelashes in affected individuals, regardless of SSV or segmental vitiligo in association with NSV after reduction of epidermal H2O2 (n=5). Taken together, our contribution strongly supports H2O2/ONOO-mediated stress in the pathogenesis of SSV. Our findings offer new treatment intervention for lost skin and hair color.—Schallreuter, K. U., Salem, M. A. E. L., Holtz, S., Panske, A. Basic evidence for epidermal H2O2/ONOO−-mediated oxidation/nitration in segmental vitiligo is supported by repigmentation of skin and eyelashes after reduction of epidermal H2O2 with topical NB-UVB-activated pseudocatalase PC-KUS.

Submitted by Diego Santos (diegosantos@unipampa.edu.br) on 2015-04-10T11:49:39Z No. of bitstreams: 1 111010003.pdf: 1306168 bytes, checksum: 65e96979f53ded7b91f629af13358a87 (MD5) / Made available in DSpace on 2015-04-10T11:49:41Z (GMT). No. of bitstreams: 1 111010003.pdf: 1306168 bytes, checksum: 65e96979f53ded7b91f629af13358a87 (MD5) Previous issue date: 2012-08-20 / O metilmercúrio (MeHg) é um agente tóxico que causa importantes prejuízos à saúde humana e ambiental. Parte desses efeitos está relacionado a sua capacidade de induzir estresse oxidativo. Os mecanismos precisos pelos quais o MeHg leva ao estresse oxidativo ainda não estão bem esclarecidos. Dados na literatura apontam para a participação de selenoproteínas como a glutationa peroxidase e a tioredoxina redutase neste processo. Neste estudo, buscou-se investigar o papel de isoformas de glutationa peroxidase (GPx1 e GPx4) e da tioredoxina redutase (TrxR1) na neurotoxicidade induzida por MeHg em camundongos, focando na atividade e expressão destas proteínas. Nossos resultados mostraram, que o tratamento de camundongos Swiss machos adultos com MeHg (40 mg/L na água de beber) durante 21 dias causa uma diminuição significativa na atividade das enzimas GPx e TrxR no córtex e cerebelo dos animais tratados, em comparação com os controles. Observou-se também uma significativa redução na expressão de GPx1, GPx4 e TrxR1 no cerebelo dos animais tratados, enquanto que no córtex apenas GPx4 e TrxR1 foram afetadas. Concomitantemente a estes resultados, observou-se um aumento significativo na atividade das enzimas antioxidantes SOD, CAT, GR e GST no cerebelo e da CAT no cortex. A expressão de HSP70 foi significativamente aumentada no cerebelo dos animais tratados. Estes dados denotam uma clara resposta celular antioxidante frente aos efeitos tóxicos do MeHg em nosso modelo, reforçando dados da literatura que indicam o estresse oxidativo como um importante mecanismos na neurotoxicidade induzida por este organometal. Além disso, nossos resultados apontam para isoformas de glutationa peroxidase e tioredoxina redutase como importantes alvos moleculares do MeHg e, ao menos, em nosso conhecimento, este é o primeiro estudo demonstrando o papel da GPx4 na neurotoxicidade induzida por este agente tóxico ambiental. Outro objetivo deste trabalho foi investigar os efeitos biológicos do extrato da alga Prasiola crispa(PcE), oriunda do continente Antártico, nos modelos de Drosophila melanogaster e Nauphoeta cinérea. Organismos adaptados a ambientes extremos como a Antártica tendem a apresentarem uma constituição única em termos de metabólitos secundários. Desta forma, estudos que visem à elucidação de efeitos biológicos de organismos oriundos destas regiões tendem a apresentarem relevância do ponto de vista biotecnológico. Nossos dados apontam para um potencial biocida de PcE nos modelos de mosca-da-fruta (Drosophila melanogaster) e barata cinerea (Nauphoeta cinerea), visto que a administração do extrato induziu toxicidade nos dois modelos. A toxicidade em D. melanogaster foi avaliada como percentagem de mortalidade, atividade locomotora (geotaxia negativa) e alterações bioquímicas incluindo atividade acetilcolinesterase (AChE) e marcadores de estresse oxidativo.Também foi investigada a ação cardiotóxica do extrato no modelo de coração semi-isolado de barata cinerea. A administração do extrato(2mg/ml) foi feita por 24 horas e, nas moscas, causou um aumento massivo na mortalidade (aumento de 7,6 vezes em relação ao controle). Também foi observado um aumento significativo na atividade locomotora, indicando uma ação neurotóxica do extrato. A atividade AChE, os níveis de glutationa e formação de hidroperóxido manteve-se inalterada. A atividade da glutationa S-transferase aumentou significativamente após a administração de PcE, já a atividade da catalase diminuiu significativamente em moscas que receberam o extrato. No modelo de coração semi-isolado de barata, foi observado uma diminuição da freqüência cardíaca. A incubação do extrato com DTNB, um forte agente oxidante, bloqueou significativamente o efeito cardiotóxico do extrato, sugerindo que compostos redutores podem ser responsáveis pelo efeito observado. Desta forma, este estudo demonstrou os efeitos tóxicos de PcE, em dois modelos de inseto, sugerindo seu potencial como bioinseticida. Os mecanismos precisos relacionados a este efeito ainda necessitam de esclarecimentos, entretanto, alterações em sistemas antioxidantes vitais podem estar envolvidos. / Methylmercury (MeHg) is a toxic agent that causes severe damage to human health and the environment. These effects are related to its ability to induce oxidative stress. The precise mechanisms by which MeHg leads to oxidative stress are not well understood. Data from literature point to the involvement of selenoproteins such as glutathione peroxidase and thioredoxin reductase in this process. In this study, we sought to investigate the role of isoforms of glutathione peroxidase (GPx4 and GPx1) and thioredoxin reductase (TrxR1) in MeHg-induced neurotoxicity in mice, focusing on activity and expression of these proteins. Our results showed that treatment of adult male mice Swiss with MeHg (40 mg / L in drinking water) for 21 days causes a significant decrease in GPx and TrxR enzyme activity in the cerebral cortex and cerebellum of treated animals when compared to controls. We also observed a significant reduction in the expression of GPx1, GPx4 and TrxR1 in the cerebellum of the treated animals, whereas in the cortex only GPx4 and TrxR1 are affected..In parallel, we observed a significant increase in antioxidant enzymes SOD, CAT, GR and GST in the cerebellum and CAT in cortex. HSP70 expression was significantly increased in the cerebellum of the treated animals. These results show a clear antioxidant cell response against the toxic effects of MeHg in our model, reinforcing the literature data indicating oxidative stress as an important mechanism in the neurotoxicity induced by this organometal. Furthermore, our results point to isoforms of glutathione peroxidase and thioredoxin reductase as important molecular targets of MeHg and, at least, to our knowledge, this is the first study demonstrating the role of GPx4 in the neurotoxicity induced by this toxic environmental agent. Another objective of this study was to investigate the biological effects of the extract of the alga Prasiola crispa (PcE), from the Antarctic continent, in the insect models Drosophila melanogaster and Nauphoeta cinerea. Organisms adapted to extreme environments such as Antarctica tend to present a unique composition in terms of secondary metabolites. Thus, studies aimed at elucidating the biological effects of organisms from these areas tend to be relevant in a biotechnological point of view. Our data demonstrates a potential biocide PcE effect in the fruit fly (Drosophila melanogaster) and lobster cockroach (Nauphoeta cinerea), since the administration of the extract induced toxicity in both models. Toxicity in D. melanogaster was assessed as percentage of mortality, locomotor activity (negative geotaxis) and biochemical measurements including acetylcholinesterase (AChE) and markers of oxidative stress. We also investigated the cardiotoxic action of the extract in a cockroach semi-isolated heart model. The administration of the extract (2 mg / ml) for 24 hours toflies, caused a massive increase in mortality (7.6-fold increase compared to control). We also observed a significant increase in locomotor activity, indicating a neurotoxic action of the extract. AChE activity, glutathione levels and the formation of hydroperoxide remained unchanged. The activity of glutathione S-transferase significantly increased after administration of PcE while catalase activity was significantly decreased in flies that received the extract. A significant decrease in heart rate in the cockroach semi-isolated heart model was observed after PcE administration. The incubation of the extract with DTNB, a strong oxidizing agent, significantly blocked the cardiotoxic effect of the extract, suggesting that reducing compounds may be responsible for the observed effect. Thus, this study demonstrated, for the first time, the toxic effects of PcE, in two insect models, suggesting its potential as a bioinsecticide. The precise mechanisms related to this effect still need clarification, however, changes in vital antioxidant systems may be involved.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / The diphenyl ditelluride (PhTe)2 is one of the most toxic organic compounds of tellurium which can make their use unsafe. The mechanism(s) involved in (PhTe)2 toxicity is(are) elusive, but thiol oxidation of critical proteins are important targets. Consequently, the possible remedy of its toxicity by thiol-containing compounds is of experimental and clinical interest. Therefore, this study aimed to evaluate the toxicity of in vivo exposure to (PhTe)2 from oxidative stress biomarkers and behavioral parameters in adult mice and the possible protective effect of N-acetylcysteine (NAC). They evaluated parameters of oxidative stress and behavior in mice. In order to alleviate the toxicity, NAC was administered before (3 days) and simultaneously (PhTe)2 (7 days). Mice were separated into six groups receiving daily injections of (1) Potassium phosphate buffer (TFK) (2.5 ml/kg, intraperitonealy (i.p.)) plus canola oil (10 ml/kg, subcutaneously (s.c.)), (2) NAC (100 mg/kg, i.p.) plus canola oil s.c., (3) TFK i.p. plus (PhTe)2 (10 μmol/kg, s.c.), (4) TFK i.p. plus (PhTe)2 (50 μmol/kg, s.c.), (5) NAC plus (PhTe)2 (10 μmol/kg, s.c.), and (6) NAC plus (PhTe)2 (50 μmol/kg, s.c.). Treatment with (PhtE) started on day 2 of treatment with NAC. The results demonstrate that (PhTe)2 induced behavioral changes in locomotor activity at a concentration of 50 μmol/kg and NAC did not change the effect of (PhTe)2. Motor coordination and lift the bar were compromised and both showed severe motor abnormalities in test animals independent of concentration of (PhTe)2 . The (PhTe)2 also inhibited important selenoenzymes, thioredoxin reductase (at concentrations of 10 μmol/kg and 50 μmol/kg) and glutathione peroxidase (at concentration of 10 μmol/kg) but produced little or no effect on the antioxidant activity of catalase and glutathione reductase. Contrary to expectation, the co-administration of NAC did not protect against deleterious effects (PhTe)2. It was possible to establish high sensitivity of brain tissue compared to the damage (PhTe)2. Other low molecular weight thiols must be investigated to determine whether they may or may not be effective against ditellurides. / O ditelureto de difenila (PhTe)2 é um dos compostos orgânicos de telúrio mais tóxicos, o que pode tornar seu emprego pouco seguro. O mecanismo envolvido na toxicidade do (PhTe)2 ainda é incerto, mas a oxidação de tióis em proteínas são alvos importantes. A partir disso, compostos contendo tiol possívelmente poderiam solucionar ou minimizar a sua toxicidade. Portanto, este estudo teve como objetivo avaliar a toxicidade da exposição in vivo ao (PhTe)2 a partir de biomarcadores de estresse oxidativo e parâmetros comportamentais em camundongos adultos e o possível efeito protetor da N-acetilcisteína (NAC). Foram avaliados parâmetros de estresse oxidativo e comportamentais em camundongos. A fim de mitigar a toxicidade, foi administrado NAC antes (3 dias) e, simultaneamente ao (PhTe)2 (7 dias). Os camundongos foram separados em seis grupos que receberam injeções diárias de (1) Tampão fosfato de potássio (TFK) (2.5 ml/kg, intraperitonealmente (i.p.)) mais óleo de canola (10 ml/kg, subcutaneamente (s.c.)), (2) NAC (100 mg/kg, i.p.) mais óleo de canola s.c., (3) TFK i.p. mais (PhTe)2 (10 μmol/kg, s.c.), (4) TFK i.p. mais (PhTe)2 (50 μmol/kg, s.c.), (5) NAC mais (PhTe)2 (10 μmol/kg, s.c.), e (6) NAC mais (PhTe)2 (50 μmol/kg, s.c.). O tratamento com (PhTe)2 começou no quarto dia de tratamento com NAC. Os resultados demonstram que (PhTe)2 induziu alterações comportamentais na atividade locomotora na concentração de 50 μmol/kg e a NAC não modificou o efeito do (PhTe)2. A coordenação motora e a força de sustentação na barra foram comprometidas e ambas revelaram alterações motoras graves nos animais testados independente da concentração de (PhTe)2. O (PhTe)2 também inibiu selenoenzimas importantes, tiorredoxina redutase (nas concentrações de 10 μmol/kg e 50 μmol/kg) e glutationa peroxidase (na concentração de 10 μmol/kg), mas produziu mínimo ou nenhum efeito sobre a atividade antioxidante da catalase e glutationa redutase. Contrariamente ao esperado, a co-administração com NAC não protegeu contra os efeitos deletérios do (PhTe)2. Foi possível estabelecer grande sensibilidade do tecido cerebral frente aos danos causados pelo (PhTe)2. Outros tióis de baixo peso molecular devem ser investigados para determinar se eles podem ou não ser eficazes contra diteluretos.

Les bactéries acidophiles du genre Acidithiobacillus joue un rôle important dans les activités industrielles de récupération des métaux au sein des sites miniers. Dans cette thèse, la séquence du génome de la bactérie psychro-tolerante Acidithiobacillus ferrivorans CF27 a été re-séquencée. L’analyse comparative du génome de CF27 et des autres bactéries du genre Acidithiobacillus a permis de montrer: (i) une synthénie conservée entre 2 clusters de tRNAs trouvés dans les génomes de At. ferrivorans CF27 et At. ferrooxidans ATCC 23270, et qui ont contribué à la redondance génique des tRNAs chez ces 2 organismes. Notre analyse in silico à grande échelle de ces clusters de tRNAs au sein des génomes procaryotes a montré que les clusters de tRNAs sont présents dans très peu de phyla bactériens; (ii) la présence d’une importante proportion de gènes spécifiques chez CF27 et SS3, ce qui indique la très grande variabilité du contenu génique dans les génomes d’Acidithiobacillus et ainsi la nature unique de chaque groupe d’espèces. L’expression de ces gènes spécifiques a été confirmée chez CF27 cultivés en présence de Fer et soufre; et (iii) une composition taxonomique chimérique des génomes de la classe des Acidithiobacillia, confirmant ainsi que ce groupe appartient à une classe taxonomique particulière. Ces résultats apporte de nouvelles connaissances sur l’adaptation de CF27 à son environnement, ainsi que la nature chimérique des génomes de la classe taxonomique Acidithiobacillia. J’ai participé au projet ‘Thioredoxine réductase (TR)’ dont l’objectif est de définir la fonction biochimique, la structure moléculaire, ainsi que l’histoire évolutive de TRi, une réductase atypique. / The acidophilic Acidithiobacillus bacteria play an important role in industrial biomining operations for metal recovery. In this thesis, the genome sequence of a psychrotolerant Acidithiobacillus ferrivorans CF27 were first refined. The comparative genome analysis between CF27 and the closely related Acidithiobacillus genomes revealed: (i) a syntenic conservation of two tRNA array units which are only present in At. ferrivorans CF27 and At. ferrooxidans ATCC 23270 genomes and mainly contribute to the tRNA gene redundancy in both organisms. Moreover, our large-scale genome survey of the tRNA array units in prokaryotic organisms showed that tRNA arrays appear in few phyla; (ii) a high proportion of species-specific genes in CF27 and SS3 strains indicated the high variability of gene content in Acidithiobacillus genomes and therefore the unique nature of each group of species. Given that mRNA expression of some CF27 specific genes were confirmed in Fe(II)-grown cells and sulfur attached cells in CF27, these results highlighted the functional importance of specific genes for CF27 lifestyle; and (iii) the mosaic taxonomic composition of members of the Acidithiobacillia class, and thus confirmed that this group belongs to a particular taxonomic class, distinct to other proteobacterial groups. Taken together, our results provide insights into At. ferrivorans lifestyle as well as the chimeric genome nature of the Acidithiobacillus organisms. In addition, I also participated to the ‘Thioredoxin reductase’ project which aims to define the biochemical function, molecular structure and evolution of TRi, an atypical thioredoxin reductase.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Lead is a heavy metal that accumulates primarily in kidney, where exerts its nephrotoxic effects. Several studies suggest that the oxidative stress is an important molecular mechanism for the toxic effects of lead in kidney and in other organs. Cytosolic thioredoxin reductase (TrxR1) is a selenoflavoprotein involved in many processes modulating intracellular reactive oxygen species levels. The aims of this study were to evaluate the effects of acute and chronic exposure to lead acetate on renal TrxR1 activity and on other oxidative stress parameters (d-aminolevulinic acid dehydratase activity, glutathione Stransferase, non-protein thiol groups, lipid peroxidation, and antioxidant enzymes in kidneys), as well as on plasmatic indicators of renal function (creatinine, uric acid and phosphate) in rats. In acute exposure, rats received a single intraperitoneal injection of 25 or 50 mg/kg lead acetate and were killed 6, 24 or 48 h later. In chronic exposure, rats received a daily intraperitoneal injection of lead acetate (5 or 25 mg/kg) during 30 days and were killed at 31st day. In our study, acute exposure to 25 mg/kg lead acetate increased superoxide dismutase (SOD) and TrxR-1 activity (after 6, 24, and 48 h), while exposure to 50 mg/kg lead acetate increased catalase (CAT) activity (after 48h) and inhibited d-aminolevulinic acid dehydratase (δ-ALA-D) activity (after 6, 24, and 48 hs) in kidneys (P < 0.05). Chronic exposure to 5 mg/kg lead acetate inhibited δ-ALA-D and increased glutathione S-transferase (GST), non protein sulfhydryl groups (NPSH), CAT, TrxR-1, and uric acid plasma levels, while exposure to 25 mg/kg lead acetate reduced body weight and δ -ALA-D, but increased GST, NPSH, and uric acid plasma levels (P < 0.05). No changes were observed in thiobarbituric acid reactive substances, glutathione peroxidase, creatinine or inorganic phosphate levels after either acute or chronic exposure. In conclusion, lead exposure caused a marked increase in the TrxR1 activity in the kidney of rats and this change may be an early indicator of acute exposure to low lead doses. However, further studies are needed to clarify the biological meaning of this induction as well as the mechanism involved in such effect. / O chumbo é um metal pesado que acumula-se preferencialmente nos rins, onde exerce seus efeitos nefrotóxicos. Muitos estudos sugerem que o estresse oxidativo seja um importante mecanismo molecular para os efeitos tóxicos do chumbo no rim e em outros órgãos. A tioredoxina redutase citosólica (TrxR1) é uma selenoflavoproteína envolvida em muitos processos reguladores dos níveis intracelulares de espécies reativas de oxigênio. Os objetivos deste estudo foram avaliar os efeitos da exposição aguda e crônica ao acetato de chumbo sobre a atividade da TrxR1 renal e sobre outros parâmetros de estresse oxidativo (atividade da δ-aminolevulinato desidratase, glutationa S-transferase, grupos tiólicos nãoprotéicos, peroxidação lipídica e enzimas antioxidantes nos rins), bem como sobre os indicadores plasmáticos da função renal (creatinina, ácido úrico e fosfato) em ratos. Na exposição aguda, os ratos receberam uma única injeção intraperitoneal de 25 ou 50 mg/kg de acetato de chumbo e foram mortos 6, 24 ou 48 horas mais tarde. Na exposição crônica, os ratos receberam uma injeção intraperitoneal diária de acetato de chumbo (5 ou 25 mg/kg) durante 30 dias e foram mortos no 31° dia. Em nosso estudo, a exposição aguda a 25 mg/kg de acetato de chumbo aumentou a atividade da superóxido dismutase (SOD) e da TrxR1 (após 6, 24 e 48 h), enquanto que a exposição a 50 mg/kg de acetato de chumbo aumentou a atividade da catalase (CAT) (após 48 h) e inibiu a atividade da δ-aminolevulinato desidratase (δ-ALA-D) (após 6, 24, 48 h) nos rins (p<0,05). A exposição crônica a 5 mg/kg de acetato de chumbo inibiu a δ-ALA-D e aumentou a glutationa S-transferase (GST), níveis de grupos tiólicos não-protéicos (SHNP), CAT, TrxR1 e níveis plasmáticos de ácido úrico (p<0,05), enquanto que a exposição a 25 mg/kg de acetato de chumbo reduziu o peso corporal e a δ-ALA-D, mas aumentou a GST, SHNP e os níveis plasmáticos de ácido de ácido úrico (p<0,05). Não houve alterações nos níveis de substâncias reativas ao ácido tiobarbitúrico (TBARS), na atividade da glutationa peroxidase (GPx) e nos níveis plasmáticos de creatinina e fosfato inorgânico tanto após a exposição aguda como após a exposição crônica. Conclui-se que a exposição ao chumbo causou um aumento significativo na atividade da TrxR1 renal de ratos e esta alteração pode ser um indicador primário da exposição aguda a baixas doses de chumbo. Entretanto, será necessária a realização de mais estudos para elucidar o significado biológico desta indução, bem como o mecanismo envolvido em tal efeito.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Oxidative stress is an important molecular mechanism of lead (Pb) toxicity. The thioredoxin system (selenoenzyme thioredoxin reductase-TrxR, thioredoxin protein- Trx and NADPH) is essential for the antioxidant defense and cellular redox control. In our previous study, it was showed that cytosolic renal TrxR1 activity of rats increased after acute and long-term exposure to Pb and this was the only parameter that changed after both exposures to low Pb doses. Then, it was suggested that TrxR1 could operate in the early defense against Pb toxicity and it could also be used as a bioindicator of early effects of Pb. Thus, the main objective of this thesis was to investigate the role of thioredoxin system in Pb-induced changes, evaluating: I) in vitro the activity of purified TrxR1, as well as the activity and the protein expression of TrxR1 and Trx1 in renal HEK 293 culture cells exposed to Pb; II) in vivo, the effects of Pb exposure in rats and in occupationally-exposed humans on renal (only in rats) and blood TrxR1 activity (both rats and humans), comparing these effects to oxidative stress parameters, as well as to classical bioindicators of Pb effect and exposure. The results of the in vitro study showed that lead is a less potent inhibitor of the purified TrxR1 activity (IC50 = 0.27 TM) than its structural homologous glutathione reductase (IC50 = 0.048 TM). TrxR1 inhibition was independent on the selenocysteine residue of the active site and was reversible by bovine serum albumin and by the EDTA chelating. TrxR1 inhibition also occurred in HEK 293 cells exposed to the highest Pb acetate concentration (60 TM), without alterations in protein expression. However, under glutathione (GSH) depletion after pre-incubation of cells with L-buthionine-[S,R]-sulfoximine (BSO) and further exposure to Pb, the activity and expression of both TrxR1 and Trx1 increased in the absence of cytotoxicity and of changes in GR and glutathione S-transferase activities, which indicates Trx system as an important protective mechanism against Pb toxic effects in GSH-depleted cells. On the other hand, blood TrxR1 activity did not change either after acute exposure of rats or long-term exposure of humans to Pb. However, the increase of renal TrxR1 activity in rats exposed to the highest dose of Pb acetate (25 mg/kg) occurred concomitantly with the increase of blood and renal Pb levels over time (6, 24 e 48 h), whereas the erythrocyte δ-ALA-D inhibition, which is a classical indicator of Pb effects, occurred after 6 h of exposure and the activity was further recovered (at 24 and 48 h). Moreover, the increase of renal TrxR1 activity occurred without renal histopathologycal damage, which corroborates the increase of this enzyme as an early event of Pb toxicity. Overall, the results of the current study point out the thioredoxin system as a target for Pb, but mainly as a protective mechanism against Pb toxicity. However, the absence of changes in blood TrxR1 activity in Pb-exposed animals and humans indicates that this enzyme is not an appropriate bioindicator of the toxic effects of Pb in exposed populations. / O estresse oxidativo é um importante mecanismo molecular da toxicidade do chumbo (Pb). O sistema da tiorredoxina (selenoenzima tiorredoxina redutase -TrxR, proteína tiorredoxina -Trx e NADPH) é essencial na defesa antioxidante e no controle redox celular. Em nosso estudo prévio, foi demonstrado que a atividade da enzima TrxR1 (citosólica) renal de ratos aumentou na exposição aguda e prolongada ao Pb, sendo o único parâmetro alterado em ambas exposições a doses baixas de Pb. Assim, foi sugerido que a TrxR1 atuaria precocemente na defesa contra a toxicidade do metal, podendo também ser utilizada como um bioindicador dos efeitos precoces do Pb. Assim, o objetivo geral desta tese foi investigar o papel do sistema da tiorredoxina nas alterações induzidas pelo Pb, avaliando: I) in vitro a atividade da TrxR1 purificada, bem como a atividade e expressão protéica da TrxR1 e Trx1 em culturas de células renais HEK 293 expostas ao Pb e II) in vivo, os efeitos do Pb em ratos e em humanos ocupacionalmente expostos ao Pb sobre a atividade da TrxR1 renal (somente em ratos) e sanguínea (ratos e humanos), comparando esses efeitos com parâmetros de estresse oxidativo, bem como com indicadores clássicos de efeito e de exposição ao Pb. Os resultados do estudo in vitro mostraram que a atividade da enzima TrxR1 purificada foi inibida pelo Pb (IC50 = 0.27 TM) de forma menos potente que a sua homóloga estrutural glutationa redutase (IC50 = 0.048 TM). Essa inibição foi independente do resíduo de selenocisteína do sítio ativo da TrxR1 e foi revertida pela albumina sérica bovina e pelo quelante EDTA. A inibição da TrxR1 também ocorreu em células HEK 293 expostas à maior concentração de acetato de Pb (60 TM), sem alterações na expressão protéica. Entretanto, quando os níveis celulares de glutationa (GSH) foram depletados por pré incubação das células com L-butionina-[S,R]-sulfoximina (BSO) e posterior exposição ao Pb, a atividade e a expressão da TrxR1 e da Trx1 aumentaram na ausência de citotoxicidade e de alterações nas atividades da GR e glutationa S-transferase, apontando esse sistema como um importante mecanismo contra a toxicidade do Pb em células sob depleção de GSH. Por outro lado, a atividade da TrxR1 sanguínea não alterou na exposição aguda de ratos e prolongada de humanos ao Pb. No entanto, o aumento da atividade da TrxR1 renal em ratos expostos à maior dose de acetato de Pb (25 mg/kg) foi concomitante com o aumento dos níveis sanguíneos e renais de Pb ao longo do tempo (6, 24 e 48 h), enquanto que a inibição da enzima δ-ALA-D eritrocitária, um indicador clássico de efeito do Pb, ocorreu após 6 h de exposição, sendo sua atividade restabelecida posteriormente (24 e 48 h). Além disso, o aumento da atividade da TrxR1 renal ocorreu sem danos histopatológicos renais, confirmando essa alteração como um evento precoce da toxicidade do Pb. Em geral, os resultados do presente estudo apontam o sistema da tiorredoxina como alvo do Pb, mas principalmente como um mecanismo de proteção contra o metal. Entretanto, a ausência de alterações na atividade da TrxR1 sanguínea em animais e humanos expostos ao Pb, indica que essa enzima não é um bioindicador adequado dos efeitos tóxicos do Pb em populações expostas.

Exposure to toxic bile acids (BA) and retinoic acids (RA) is implicated in toxicities related to excessive oxidative stress. This thesis examined roles and mechanisms of the oxidative stress-responsive nuclear factor (erythroid 2-like) factor 2 (Nrf2) in adaptive cell defense against BA and RA toxicities. Using liver cells and mouse models, many antioxidant proteins known to be Nrf2 target genes, particularly the rate-limiting enzyme for glutathione (GSH) biosynthesis, i.e., glutamate-cysteine ligase subunits (GCLM/GCLC), were induced by BA [lithocholic acid (LCA)] or RA (all-trans, 9-cis and 13-cis) treatment. Evidence for increased Nrf2 transactivation by LCA and all-trans-RA was exemplified in HepG2 by: (1) reduced constitutive and inducible expression of GCLM/GCLC upon Nrf2 silencing via small-interfering RNA; (2) increased inducible expression of GCLM/GCLC genes by Nrf2 overexpression, but overexpression of dominant-negative Nrf2 decreased it; (3) increased nuclear accumulation of Nrf2 as signature event of receptor activation; (4) enhanced Nrf2-dependent antioxidant-response-element (ARE) reporter activity as indicative of increased Nrf2 transactivation; and (5) increased Nrf2 occupancy to AREs of GCLM and GCLC. Additionally, in BA-treated HepG2 cells, we observed concomitant increases of many ATP-binding cassette (ABC) transporters (MRPs 1-5, MDR1 and BCRP) in parallel with increased cellular efflux. Nrf2 silencing in HepG2 cells decreased constitutive and inducible expression of MRP2, MRP3 and ABCG2. However, Nrf2-silenced mouse hepatoma cells, Hepa1c1c7, and Nrf2-/- mice had decreased constitutive and/or inducible expression of Mrps 1-4, suggesting species differences in Nrf2-dependent regulation of hepatic ABC transporters. Protection by Nrf2 against BA and RA toxicities was confirmed by observations that Nrf2 silencing increased cell susceptibility to BA- and RA-induced cell death. Moreover, Nrf2-/- mice suffered more severe liver injury than the wildtype. Increased GSH and efflux activity following increased GCLM/GCLC and ABC transporters, respectively, can mitigate LCA toxicity. Activation of MEK1-ERK1/2 MAPK was shown to primarily mediate Nrf2 transactivation and LCA-induced expression of antioxidant proteins and Nrf2-dependent and -independent ABC transporters. In conclusion, Nrf2 activation by BA and RA led to coordinated induction of antioxidant and ABC proteins, thereby counteracting resultant oxidative cytotoxicity. The potential of targeting Nrf2 in management of BA and RA toxicities merits further investigation.

Exposure to toxic bile acids (BA) and retinoic acids (RA) is implicated in toxicities related to excessive oxidative stress. This thesis examined roles and mechanisms of the oxidative stress-responsive nuclear factor (erythroid 2-like) factor 2 (Nrf2) in adaptive cell defense against BA and RA toxicities. Using liver cells and mouse models, many antioxidant proteins known to be Nrf2 target genes, particularly the rate-limiting enzyme for glutathione (GSH) biosynthesis, i.e., glutamate-cysteine ligase subunits (GCLM/GCLC), were induced by BA [lithocholic acid (LCA)] or RA (all-trans, 9-cis and 13-cis) treatment. Evidence for increased Nrf2 transactivation by LCA and all-trans-RA was exemplified in HepG2 by: (1) reduced constitutive and inducible expression of GCLM/GCLC upon Nrf2 silencing via small-interfering RNA; (2) increased inducible expression of GCLM/GCLC genes by Nrf2 overexpression, but overexpression of dominant-negative Nrf2 decreased it; (3) increased nuclear accumulation of Nrf2 as signature event of receptor activation; (4) enhanced Nrf2-dependent antioxidant-response-element (ARE) reporter activity as indicative of increased Nrf2 transactivation; and (5) increased Nrf2 occupancy to AREs of GCLM and GCLC. Additionally, in BA-treated HepG2 cells, we observed concomitant increases of many ATP-binding cassette (ABC) transporters (MRPs 1-5, MDR1 and BCRP) in parallel with increased cellular efflux. Nrf2 silencing in HepG2 cells decreased constitutive and inducible expression of MRP2, MRP3 and ABCG2. However, Nrf2-silenced mouse hepatoma cells, Hepa1c1c7, and Nrf2-/- mice had decreased constitutive and/or inducible expression of Mrps 1-4, suggesting species differences in Nrf2-dependent regulation of hepatic ABC transporters. Protection by Nrf2 against BA and RA toxicities was confirmed by observations that Nrf2 silencing increased cell susceptibility to BA- and RA-induced cell death. Moreover, Nrf2-/- mice suffered more severe liver injury than the wildtype. Increased GSH and efflux activity following increased GCLM/GCLC and ABC transporters, respectively, can mitigate LCA toxicity. Activation of MEK1-ERK1/2 MAPK was shown to primarily mediate Nrf2 transactivation and LCA-induced expression of antioxidant proteins and Nrf2-dependent and -independent ABC transporters. In conclusion, Nrf2 activation by BA and RA led to coordinated induction of antioxidant and ABC proteins, thereby counteracting resultant oxidative cytotoxicity. The potential of targeting Nrf2 in management of BA and RA toxicities merits further investigation.