Orientadores: Mônica Barbosa de Melo, Fernando Ferreira Costa / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-21T00:19:46Z (GMT). No. of bitstreams: 1 Gil_GislenePereira_M.pdf: 2808239 bytes, checksum: 22a11f67b6d086bf9bab0100ff802ec7 (MD5) Previous issue date: 2012 / Resumo: Embora a anemia falciforme (AF) resulte da homozigosidade de uma única mutação, no codon 6 do locus da ?-globina, fenotipicamente, essa doença é muito heterogênea, de modo que diferentes pacientes podem apresentar evoluções clínicas significativamente distintas. As complicações nestes pacientes normalmente são decorrentes de acometimento vascular causado pelo acúmulo de hemácias falcizadas nos vasos sanguíneos. Um dos eventos que vem sendo associados a complicações em diversas doenças é o mecanismo de estresse oxidativo, o qual apresenta- se exacerbado em pacientes com AF. Dentre as fontes de estresse oxidativo nestes pacientes estão os eventos de vaso-oclusão e isquemia reperfusão, os quais são muito frequentes. O estresse oxidativo em níveis elevados pode danificar várias moléculas e posteriormente prejudicar o organismo. Alguns polimorfismos em enzimas envolvidas na via de estresse oxidativo foram associados com doenças vasculares como, hipertensão, doença arterial coronária, doença arterial periférica. Considerando que os pacientes com AF apresentam complicações decorrentes de acometimento vascular, esses polimorfismos podem estar contribuindo para as várias manifestações clínicas e, consequentemente, para a gravidade da doença. Este projeto se propôs a avaliar três polimorfismos em genes envolvidos no mecanismo de estresse oxidativo em pacientes com AF e testar a associação com o grau de gravidade da doença. Os polimorfismos analisados foram o C242T e -930 A/G no gene CYBA e o -413 T/A no gene HMOX-1. Os genótipos foram identificados por meio das técnicas de PCR e sequenciamento direto, e os escores de gravidade foram obtidos através de índices de severidade, em 169 pacientes. O genótipo AA do polimorfismo -930 A/G apresentou-se associado com escores baixos de gravidade, obtidos através do índice pediátrico, nas crianças com anemia falciforme. O genótipo CT do polimorfismo C242T esteve associado com crises álgicas e o genótipo TT do polimorfismo -413 T/A mostrou-se associado a níveis elevados de HbF, através de análises dos dados de todos os pacientes. De acordo com este estudo, sugerimos novos marcadores genéticos, os quais podem estar direta ou indiretamente envolvidos com a gravidade da doença em pacientes com AF na população brasileira. Estudos futuros em grandes coortes necessitam ser realizados para confirmar esses resultados / Abstract: Although sickle cell anemia (SCA) results from the homozygosity for a single mutation at codon 6 of the ?-globin locus, phenotypically this disease is very heterogeneous, so that different patients may have significantly different clinical outcomes. The complications in these patients are usually caused by vascular impairment caused by the accumulation of sickled erythrocytes in the blood vessels. One event that has been associated with complications in several diseases is oxidative stress, which has increased levels in SCA patients. Among the sources of oxidative stress in these patients are the vaso-occlusion and ischemia reperfusion events, which are very common. Oxidative stress at high levels can damage various molecules and subsequently damage the organism. Some polymorphisms of enzymes involved in the oxidative stress pathway have been associated with vascular diseases as: hypertension, coronary heart disease, peripheral arterial disease. Considering that the complications in SCA patients are due to vascular involvement, these polymorphisms may be contributing to the various clinical manifestations and consequently to the severity of the disease. This project proposes to evaluate three polymorphisms in genes involved in the mechanism of oxidative stress in SCA patients and test the association with the severity of the disease. The analyzed polymorphisms were C242T and -930 A/G in the CYBA gene and the -413 T/A in the HMOX-1 gene. The genotypes were identified through PCR and direct sequencing, and severity scores were obtained by severity indexes in 169 patients. The AA genotype of the -930 A/G polymorphism was associated with low severity scores, obtained from the pediatric index, in children with sickle cell anemia. The CT genotype of the C242T polymorphism was associated with pain crisis and the TT genotype of the -413 T/A polymorphism was associated with high levels of HbF, after analyzing the data from all patients. According to this study, new genetic markers can be suggested, which may be, directly or indirectly, involved with disease severity in patients with SCA in the Brazilian population. Future studies in larger cohorts have to be conducted to confirm these results / Mestrado / Clinica Medica / Mestre em Clinica Medica

Primary chick embryo liver cells were used to explore the regulation of δ-aminolevulinic acid synthase and heme oxygenase, the enzymes that catalyze the rate-limiting reactions of heme anabolism and catabolism, respectively. The general focus of the work was the exploration of the novel observation in which glutethimide and iron synergistically induced both δ-aminolevulinic acid synthase and heme oxygenase, a phenomenon that would not be predicted a priori. The course of events appeared to be: first, that heme synthesis was increased after addition of the glutethimide and that iron potentiated heme synthesis; second, the heme induced heme oxygenase five to ten fold; and third, that heme oxygenase degraded the heme permitting an uncontrolled induction of δ-aminolevulinic acid synthase. This induction of δ-aminolevulinic acid synthase could be prevented by the addition of a metalloporphyrin inhibitor of heme oxygenase. Induced δ-aminolevulinic acid synthase activity could be dramatically reduced by the addition of nanomolar concentrations of a metalloporphyrin, inhibitory for heme oxygenase, and heme. Specific observations related to the synergistic induction of heme oxygenase by glutethimide and iron was that the induction of heme oxygenase activity by glutethimide and iron occurred rapidly, with maximal increases occurring four to six hours after original treatment. Induction of heme oxygenase by glutethimide and iron was shown to be dependent on de novoheme synthesis since 4,6-dioxoheptanoic acid, a potent and specific inhibitor of heme biosynthesis, prevented the activity of heme oxygenase from increasing in the presence of glutethimide and iron. Induction of activity was associated with increases in heme oxygenase mRNA and protein; and, when induction was prevented by 4,6-dioxoheptanoic acid, no increase in either mRNA or immunoreactive protein was observed. δ-Aminolevulinic acid synthase activity was also synergistically increased by glutethimide and iron; this increase occurred 4-6 hours after maximal heme oxygenase activity had been attained. The temporal relationship between the induction of δ-aminolevulinic acid synthase and heme oxygenase suggested that the oxygenase depleted a regulatory heme pool that would normally prevent uncontrolled induction of the synthase. When cultures were exposed to tin-mesoporphyrin, a potent inhibitor of heme oxygenase, induction of δ-aminolevulinic acid synthase, normally produced by glutethimide and iron, was prevented. Addition of tin-mesoporphyrin after δ-aminolevulinic acid synthase induction had already been established promptly halted any further induction. When heme or a combination of heme and tin-mesoporphyrin was added after induction of δ-aminolevulinic acid synthase was established, activity of the synthase was rapidly reduced. Finally, experiments in primary chick embryo liver cells with tin-, zinc- and copper- chelated porphyrins were done to assess their effects on activities of δ-aminolevulinic acid synthase, induced by prior treatment of cells with glutethimide and iron. Nanomolar concentrations of zinc- or tin porphyrins reduced δ-aminolevulinic acid synthase activities, while copper-chelated porphyrins did not. When nanomolar concentrations of heme were added with zinc- or tin-porphyrins, δ-aminolevulinic acid synthase activity was further reduced. Effects of the non-heme metalloporphyrins on δ-aminolevulinic acid synthase were closely correlated with their abilities to inhibit heme oxygenase (r=0.78). The largest decrease of δ-aminolevulinic acid synthase (67%) was obtained with zinc-mesoporphyrin and heme. There was a rapid appearance of the cytosolic, precursor form of δ-aminolevulinic acid synthase in the presence of both 10 μM heme or 50 nM zinc-mesoporphyrin and 200 nM heme. Reduction of the half-life of the mRNA from 5.2 hours to 2.2-2.5 hours was observed in the presence of both 10 μM heme or 50 nM zinc-mesoporphyrin and 200 nM heme. In summary, the chick embryo liver cell culture model treated with glutethimide and iron may serve as one experimental model for patients suffering from acute porphyrias, in whom uncontrolled induction of hepatic δ-aminolevulinic acid synthase plays a key role in pathogenesis of disease. The synergistic induction of δ-aminolevulinic acid synthase in the presence of glutethimide and iron may serve as an experimental paradigm for this disease. The reduction of δ-aminolevulinic acid synthase by low doses of zinc-mesoporphyrin and heme may help form the experimental foundation for eventual studies in patients suffering from acute porphyrias.

Orientador: Stephen Hyslo / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-11T19:41:01Z (GMT). No. of bitstreams: 1 Bifaroni_RenataManoScatamburlo_M.pdf: 12304500 bytes, checksum: a5c3c67724b7ea654b79231b9f69925a (MD5) Previous issue date: 2008 / Resumo: A heme oxigenase (HO) é uma enzima envolvida na degradação de grupos heme, levando à formação de biliverdina IX, ferro e monóxido de carbono. Em baixas concentrações, esses produtos exercem diversos efeitos protetores, inclusive antiapoptóticos, antiinflamatórios, antiproliferativos e antitrombóticos, enquanto, em concentrações elevadas, podem resultar em hipóxia, comprometimento neurológico, disfunções mitocondriais e hiperbilirrubinemia. Neste estudo, investigamos o envolvimento da HO no aumento da permeabilidade vascular, na hemorragia e na mionecrose causadas pela peçonha de Bothrops jararacussu. A peçonha de B. jararacussu (0,3-3 µg/sítio) aumentou a permeabilidade vascular no dorso de pele de camundongos. Este aumento foi inibido pelo zinco deuteroporfirina 2,4-bis-glicol (ZnDPBG), um inibidor não seletivo da HO, quando administrado i.p. (45 e 90 µmol/kg), mas não quando i.d. (25-200 nmol/sítio). O co-tratamento com o inibidor do óxido nítrico sintase, N?-nitro-L-arginina metil éster (L-NAME, 100 nmol/sítio), não potencializou a inibição por ZnDPBG. ZnDPBG (45 ou 90 µmol/kg, i.p.) não reduziu a hemorragia na pele (3 e 10 µg de peçonha/sítio), mas aumentou esta resposta na dose maior. A peçonha (25 µg e 75 µg) aumentou a atividade da HO em músculo gastrocnêmio de camundongos em dois momentos - um precoce (nas primeiras horas após o envenenamento - fase de lesão aguda) e um tardio (alguns dias após a injeção de peçonha, fase de regeneração muscular). Na dose de 75 µg da peçonha, ocorreu aumento da expressão gênica da HO-1, entre 3 e 12 horas após o envenenamento, com pouca alteração na expressão da HO-2. As alterações histológicas causadas pela peçonha (25 µg e 75 µg) consistiram de três fases: fase 1 (até 6 h ou 12 h pós-peçonha - p.p.), caracterizada por hemorragia e mionecrose, fase 2 (de 12 h a 72 h p.p.), caracterizada por um extenso infiltrado inflamatório e fase 3 (7-28 dias p.p.), caracterizada por células regenerativas e maior deposição de colágeno ao redor destas células. O tratamento com ZnDPBG (90 µmol/kg, i.p.), 15 minutos após a injeção da peçonha (75 µg), inibiu parcialmente a mionecrose, diminuindo também os níveis de CK nas primeiras horas (1-3 h) pós-peçonha. Estes resultados indicam um envolvimento da HO no edema e na mionecrose causadas pela peçonha de B. jararacussu, e mostram que a inibição desta atividade com ZnDPBG pode proporcionar alguma proteção contra esses efeitos locais / Abstract: Heme-oxygenase (HO) is an enzyme involved in the degradation of heme groups that results in the formation of biliverdin IX, iron and carbon monoxide. At low concentrations, these reaction products have anti-apoptotic, anti-inflammatory, anti-proliferative, and anti-thrombotic activities, whereas at high concentrations they can cause hypoxia, neurological damage, mitochondrial dysfunction and hiperbilirrubinemia. In this study, we investigated the involvement of HO in the increase in vascular permeability, hemorrhage and myonecrosis caused by Bothrops jararacussu snake venom. Bothrops jararacussu venom (0.3-3 µg/site) increased the vascular permeability in mouse dorsal skin. This increase was inhibited by zinc deuteroporphyrin 2,4-bis-glycol (ZnDPBG), a non-selective HO inhibitor, when administered i.p. (45 and 90 µmol/kg). In contrast, ZnDPBG given i.d. (25-200 µmol/site) did not affect the increase in vascular permeability. Co-treatment with the nitric oxide synthase inhibitor N?-nitro-L-arginine methyl ester (L-NAME, 100 nmol/site) did not potentiate the inhibition by ZnDPBG. ZnDPBG (45 or 90 µmol/kg, i.p.) did not reduce the dorsal skin hemorrhage (3 and 10 µg of venom/site), but rather, increased this response at the highest dose. Venom (25 and 75µg) increased the HO activity of mouse gastrocnemius muscle. The increase in activity was biphasic and included early (first few hours after envenoming, corresponding to acute damage) and late (7-18 days after envenoming - muscle regeneration) phases. Enhanced gene expression of HO-1 was seen 3-12 h after envenomation (75 µg), with little change in HO-2 expression. Histological analysis showed that the muscle damage consisted of three phases: phase 1 (up to 6-12 h post-venom), characterized mostly by hemorrhage and myonecrosis, phase 2 (12-72 h post-venom), characterized by an extensive inflammatory infiltrate, and phase 3 (7-28 days post-venom), characterized mainly by regenerative cells and an increase in collagen deposition around these cells. Treatment with ZnDPBG (90 µmol/kg, i.p.) 15 min after venom (75 µg) injection partially attenuated the myonecrosis and also decreased the creatine kinase (CK) levels after 1 and 3 h. Together, these results indicate that HO activity is involved in the edema and myonecrosis caused by B. jararacussu venom, and that the inhibition of this activity with ZnDPBG provides some protection against these local effects / Mestrado / Mestre em Farmacologia

The chicken hepatoma cell line, LMH, was evaluated with respect to its usefulness for studies of the regulation of heme metabolism. Levels of δ-aminolevulinate synthase mRNA arid accumulation of porphyrins were used to evaluate the heme biosynthetic pathway. Regulation of heme oxygenase-1 by known inducers was used as a measure of heme degradation. The induction of heme oxygenase-1 by sodium arsenite was characterized. AP-1 transcription factor elements and MAP kinase signal transduction pathways that modulate expression of endogenous heme oxygenase-1 and transfected heme oxygenase-1 reporter gene constructs in response to arsenite were delineated. In initial studies, the drug glutethimide was used alone or in combination with ferric nitrilotriacetate to induce δ-aminolevulinate synthase mRNA. Levels of porphyrins, intermediates in the heme biosynthetic pathway, and levels of δ-aminolevulinate synthase mRNA were increased by these treatments in a manner similar to those previously observed in the widely used model system, primary chick embryo liver cells. The iron chelator, deferoxamine, gave a characteristic shift in the glutethimide induced porphyrin accumulation in primary hepatocytes, but was found to have no, effect on LMH cells. Heme mediated repression of δ-aminolevulinate synthase mRNA levels was similar among primary hepatocytes and LMH cells. Heme oxygenase-1 was regulated by heme, metals, heat shock, and oxidative stress-inducing chemicals in LMH cells. Heat shock induction of heme oxygenase-1 mRNA levels was observed for the first time in primary chick embryo liver cells. These data supported the further use of LMH cells to elucidate mechanisms responsible for modulating heme oxygenase-1 gene expression in response to inducers. The remainder of the studies focused on the role of heme oxygenase-1 as a stress response protein. The oxidative stress inducer, sodium arsenite was used to probe the cellular mechanisms that control the expression of heme oxygenase-1. A series of promoter-reporter constructs were used to search the heme oxygenase-1 promoter for arsenite responsive elements. Several activator protein-1 (AP-1) transcription factor binding elements were identified by computer sequence analysis. Three of these sites, located at -1578, -3656, and -4597 base pairs upstream of the transcription start site, were mutated. The arsenite responsiveness of the reporter constructs containing mutated AP-1 elements was less than that of the same constructs containing wild type AP-1 elements. At least part of the arsenite-mediated induction of heme oxygenase-1 required the activity of AP-1 transcriptional elements. The MAP kinase signal transduction pathways and heme oxygenase-1 are activated by similar stimuli, including cellular stress. MAP kinases have been shown to exert control over gene expression through effects on the AP-1 family of transcription factors. The MAP kinases ERK, JNK, and p38 were activated by arsenite in LMH cells. Constitutively activated components of the ERK and p38 pathways increased expression of heme oxygenase-1 promoter-luciferase reporter constructs. Arsenite-mediated induction of heme oxygenase-1 was blocked by dominant negative ERK or p38 pathway components, and by specific inhibitors of MEK (upstream ERK kinase) or p38. In contrast, reporter gene expression was unchanged in the presence of constitutively activated JNK pathway components. Dominant negative JNK pathway components had no effect on arsenite induced heme oxygenase-1 gene activity. In summary, LMH cells were characterized as a new model system for the study of heme metabolism. This cell line was then used to delineate promoter elements and signaling pathways involved in the arsenite responsiveness of heme oxygenase-1 gene expression. Three AP-1 transcription factor binding sites in the heme oxygenase-1 promoter region were required for responsiveness to arsenite. The MAP kinases ERK and p38 were shown to play an integral role in arsenite-mediated induction of heme oxygenase-1. These studies elucidate one facet of heme oxygenase-1 regulation, and provide tools that will be useful in delineating additional regulatory mechanisms.