Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 5 uppsatser.

Thesis (MSc (Genetics))--University of Stellenbosch, 2007. / The results of pharmacotherapy can vary both within and between different populations and ethnic groups. Although numerous factors are believed responsible for observed discrepancies in drug response, genetic differences, most often in the form of single nucleotide polymorphisms (SNPs), between individuals and ethnic groups are an important and at times predominant factor. The response to antiretroviral (ARV) drugs for the treatment of human immunodeficiency virus (HIV)-infection is not dissimilar. Marked variations in both ARV efficacy and occurrence of adverse drug reactions (ADRs) have been observed on both an individual and ethnic group level, which are largely attributed to polymorphisms within genes involved in the metabolism and transport of these compounds – such genes include the CYP2B6 and CYP3A4 genes, both members of the cytochrome P450 (CYP) gene superfamily, and the multidrug-resistance 1 (MDR1) gene encoding an efflux transporter protein, phosphoglycoprotein (PGP). An improved understanding of the genetic influences on ARV drug response could lead to improved therapies with fewer side-effects and minimised drug resistance. The main aim of this study was thus to investigate the genetic basis of observed differences in ARV therapy (ART) response in South African ethnic groups. Deoxyribonucleic acid (DNA) samples were collected from 206 HIV-positive individuals of Mixed-Ancestry and Xhosa ethnicity that were currently or prospectively receiving ART. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis was employed to screen the A-392G SNP in CYP3A4, the G516T and A785G SNPs in CYP2B6, and the T-129C, C1236T, G2677T/A and C3435T SNPs in MDR1. Hardy-Weinberg equilibrium (HWE) and haplotype analyses were subsequently performed on the resultant SNP genotype and ...

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The Pampa Biome has a great biodiversity of plants, among them the Family Malvaceae. Despite the medicinal use of many of these plants, studies on the biological activity of these are still very scarce. This is the case of the plant known as "erva de ovelha", which is traditionally used in therapy against prostate cancer, used as tea and prepared from two species of the genus Pavonia, highlighting the Pavonia xanthogloea. Therefore, this study aimed to identify bioactive compounds in the extract and five fractions of P. xanthogloea, evaluating their antioxidant and antitumor action in the lineage of prostate cancer DU145. Additionally, the potential cytotoxic effect via pharmacogenetic studies on human red blood cells (RBCs) bearers of different genotypes of the polymorphism found in the gene of the enzyme superoxide dismutase manganese dependent (Ala16Val-SOD2). These RBCs afterwards were exposed to extract and fractions of P. xanthogloea. The results were arranged and shown as three scientific papers. Manuscript 1: The chemical composition of the extract and fractions (ethyl acetate, hexane, n-butanol, aqueous and dichloromethane) was determined by liquid chromatography of high efficiency. The antioxidant activity was determined by DPPH assay. The antioxidant activity and the cytotoxicity evaluated by exposure of human lymphocytes exposed to different concentrations of extract/fractions with and without sodium nitroprusside and H2O2. Tiliroside was found in all extracts. The aqueous and ethyl acetate fractions showed high antioxidant capacity. The crude extract (ethanol) and hexane, n-butanol and aqueous fractions reverts the ROS levels generated by H2O2. The crude extract and hexane, ethyl acetate and n-butanol fractions do not cause cytotoxicity. The aqueous fraction was cytotoxic at concentration of 300 μg/mL. The reversal of the cytotoxicity caused by SNP was dependent on the concentration and extract/fraction. The dichloromethane fraction was cytotoxic at all concentrations. The results suggest potential medicinal use of this species. Manuscript 2: The study evaluated in vitro the hemolytic effect of chelerythrine (CHE), alkaloid extracted from Zanthoxylum rhoifolium, and the possible pharmacogenetic influence of SOD2-Ala16Val in RBCs. The CHE inhibits protein kinase C activity decreasing the RBCs deformation and causing oxidative stress. Blood was collected from healthy subjects previously genotyped for the polymorphism Ala16Val-SOD2. The CHE was incubated with RBCs (1 × 109 cell/mL) at concentrations 0.1 μM, 2 μM and 8 μM. Pharmacogenetic effect was evaluated through spectrophotometric analysis of indicators of oxidative stress, lipid peroxidation, catalase, advanced oxidation protein products (AOPP) and nitrate/nitrite (NOx), and hemolysis. The RBCs were maintained under controlled conditions. The response to treatment with CHE was genotype dependent and AA RBCs were more sensitive than VV. The SOD2 plays an important role in erythropoiesis and causes an impact on the quality of RBC. Therefore, the results presented here suggest toxicogenetic influence of SOD2 in hemolytic assay using human cells. Manuscript 3: To evaluate the antitumor effect viability assays of (24 hour exposure) and cell proliferation (72 hour exposure) were performed by MTT assay and spectrophotometric analisys. The pharmacogenetic effect through the hemolysis assay by spectrophotometry. Blood was collected from healthy subjects previously genotyped for the polymorphism Ala16Val-SOD2. Both erythrocytes and tumor cells were grown under controlled conditions. Study the pattern of hemolysis associated with different genotypes of the polymorphism Ala16Val-SOD2 suggests pharmacogenetic effect. When hemolysis assay was performed for different concentrations of extract and fractions of P. xanthogloea the anti-hemolytic protective effect observed was independent of the gene SOD2. Cytotoxic and antiproliferative activity was observed in the cell lineage of prostate cancer DU145 was dependent on the type of extract/fraction and concentration, highlighting n-butanol fraction that showed strong antiproliferative activity. These results showed great similarity to antitumor activity of doxorubicin chemotherapeutic and also seem to be associated with tiliroside isolated from P. xanthogloea. The overall results match with the popular use of P. xanthogloea regarding the prostate cancer therapy. However, further investigations should be performed to evaluate the antitumor action mechanisms of P. xanthogloea. / O Bioma Pampa possui uma grande biodiversidade de plantas, entre as quais as da Família Malvaceae. Apesar do uso medicinal de muitas destas plantas, estudos sobre a atividade biológica das mesmas ainda são muito escassos. Este é o caso da planta conhecida como erva de ovelha , que é tradicionalmente utilizada na terapia contra o câncer de próstata, na forma de chá e preparado a partir de duas espécies do gênero Pavonia, com destaque a Pavonia xanthogloea. Portanto, o presente estudo teve como objetivo principal identificar compostos bioativos do extrato e cinco frações de P. xanthogloea, avaliando a sua ação antioxidante e antitumoral na linhagem de câncer de próstata DU145. Adicionalmente, o potencial efeito citotóxico via estudos farmacogenéticos em células sanguíneas vermelhas (RBCs) humanas, portadoras de diferentes genótipos do polimorfismo encontrado no gene da enzima superóxido dismutase dependente de manganês (Ala16Val-SOD2). Estas RBCs posteriormente foram expostas ao extrato e frações da P. xanthogloea. Os resultados obtidos foram organizados e apresentados sob a forma de três artigos científicos. Manuscrito 1: A composição química do extrato e frações (acetato de etila, n-butanol, hexano, aquosa e diclorometano) foi determinada por cromatografia de líquida de alta eficiência. A capacidade antioxidante foi determinada pelo ensaio do DPPH. A atividade antioxidante e a citotoxicidade avaliada através da exposição de linfócitos humanos expostos a diferentes concentrações de extrato/frações com e sem nitroprussiato sódico e H2O2. O tilirosídeo foi encontrado em todos os extratos. As frações aquosa e acetato de etila mostraram alta capacidade antioxidaente. O extrato bruto (etanólico) e frações hexano, aquosa e n-butanol revertem os níveis de ROS gerados pelo H2O2. O extrato bruto e frações hexano, acetato de etila e n-butanol não causam citotoxidade. A fração aquosa foi citotóxica na concentração de 300 μg/mL. A reversão da citotoxicidade causada pelo SNP foi dependente do extrato/fração e da concentração. A fração diclorometano foi citotóxica em todas as concentrações. O resultado sugere potencial uso medicinal desta espécie. Manuscrito 2: O estudo avaliou in vitro o efeito hemolítico da Queleritrina (CHE), alcaloide extraído da Zanthoxylum rhoifolium, e da possível influência farmacogenética da SOD2-Ala16Val em RBCs. A CHE inibe a atividade da proteina quinase C diminuindo a deformação das RBCs e causando estresse oxidativo. O sangue foi coletado de sujeitos saudáveis, previamente genotipados para o polimorfismo Ala16Val-SOD2. A CHE foi incubada com RBCs (1 × 109 cell/mL) nas concentrações 0.1 μM, 2 μM and 8 μM. O efeito farmacogenético foi avaliado através de análises espectrofotométricas de indicadores do estresse oxidativo, lipoperoxidação, catalase, produtos avançados da oxidação de proteínas (AOPP) e nitrato/nitrito (NOx), e hemólise. As RBCs foram mantidas em condições controladas. A resposta ao tratamento com CHE foi genótipo dependente e, RBCs AA foram mais sensíveis que as VV. A SOD2 tem um importante papel na eritropoiese e causa impacto na qualidade das RBC. Portanto, os resultados aqui apresentados sugerem influência toxicogenética de SOD2 em ensaio hemolítico utilizando células humanas. Manuscrito 3: Para avaliar o efeito antitumoral foram feitos ensaios de viabilidade (24 horas de exposição) e proliferação celular (72 horas de exposição) e análise espectrofotométrica pelo ensaio MTT. O efeito farmacogenético através do ensaio de hemólise, por espectrofotometria. O sangue foi coletado de sujeitos saudáveis, previamente genotipados para o polimorfismo Ala16Val-SOD2. Tanto os eritrócitos quanto as células tumorais foram cultivadas em condições controladas. O estudo do padrão de hemólise associado aos diferentes genótipos do polimorfismo Ala16Val-SOD2 sugere efeito farmacogenético. Quando foi realizado ensaio de hemólise para diferentes concentrações do extrato e frações de P. xanthogloea o efeito anti-hemolítico protetor observado foi independente do gene da SOD2. Atividade citotóxica e antiproliferativa observada na linhagem celular de câncer de próstata DU145 foi dependente do tipo de extrato/fração e concentração, com destaque a fração butanol que apresentou intensa atividade antiproliferativa. Estes resultados apresentaram grande similaridade a atividade antitumoral do quimioterápico doxirrubicina e também parecem estar associados ao tilirosídeo, isolado da P. xanthogloea. O conjunto dos resultados condizem com o uso popular da P. xanthogloea em relação a terapia do câncer de próstata. Entretanto, investigações complementares devem ser feitas para avaliar os mecanismos de ação antitumorais da P. xanthogloea.

55 Abstract Frequency of selected genetic polymorphisms of cytochrome P450 in the Czech population and the influence of CYP2C9 genotype on the hypolipidemic effect of fluvastatin Introduction: One of the main factors of genetically determined variability in response of humans to administered drugs are differences in catalytic activity of metabolizing enzymes, which are caused mainly by genetic polymorphisms in cytochrom P450 family enzymes. This thesis consists of two parts and it is presented as a commentary to the original papers. The first aim was to investigate the frequency of functionally important variant alleles of three main isoenzymes of cytochrome P450 gene: CYP2D6, CYP2C9, CYP2C19, throughout the Czech population, predict the prevalence of poor metabolizer phenotypes, and then to compare the results to the data from other populations. Secondly, we analysed the correlation between the CYP2C9 genotype and cholesterol-lowering effect of fluvastatin in human hypercholesterolemic patients. Methods: Genotypes were determined by PCR-RFLP. The presence of alleles CYP2D6*1, *6, *5, *4, *3, and gene duplication was analysed in 233 healthy volunteers, CYP2C9*1, *2 and*3 in 254 subjects and CYP2C19*1, *2 and *2 in 218 subjects. Eighty seven patients on fluvastatin therapy, and 48 patients on monotherapy...

Frequency of occurrence of selected single nucleotide polymorphisms of CYP2C8 and MDR1 in the Czech population and their influence on the effect of amiodarone Introduction: Variability in drug response is sometimes conditioned by genetic differences in the metabolism and the transport of drugs. Interindividual differences are often caused by polymorphisms affecting biotransformation activity of enzymes and expression of transporters. In the thesis we paid attention to the cytochrome P450 CYP2C8 and MDR1. First, we described the frequency of occurrence of selected variant alleles CYP2C8 * 2, CYP2C8 * 3 (2 substitution in exon 3 and 8, CYP2C8 and CYP2C8 * 3G416A * 3A1196G), CYP2C8 * 4, CYP2C8 P404A in the healthy Czech population and MDR1 variant alleles in these exons: 26 C3435T, 21 G2677A/T, 12 C1236T a 17 T-76A. Subsequently, we studied the influence of these polymorphisms on effects of amiodarone in the selected group of patients. Methods: We determined genotypes MDR1 a CYP2C8 by PCR-RFLP by using restriction enzymes and specific primers. We determined the frequency of MDR1 genotypes in 189 healthy volunteers and CYP2C8 in 161 healthy subjects. Further we included into the study 63 patients treated with amiodarone for longer than two months. Their treatment was assessed from medical records and...

NADPH-P450 oxidoreductase (POR) is a membrane bound flavoprotein that donates electrons to a wide spectrum of heme-containing proteins, among which are several steroidogenic and many xenobiotics-metabolizing enzymes. Given the important role of POR protein in drug metabolism and pharmacogenomics, there is a particular need to understand the contributions of POR genetic variants to these processes. Mutations in POR gene cause a disorder called POR deficiency, which manifests with a wide phenotypic spectrum ranging from disordered steroidogenesis to skeletal malformation, namely, Antley-Bixler syndrome (ABS). The aim of the present work was to investigate the POR gene in patients suspected to have POR deficiency syndrome from Czech Republic and to perform genotyping in Czech and Jewish control populations. We analyzed 644 alleles in unrelated individuals from the general Czech population and 1128 alleles in Jewish population, where 330 alleles were of Askhenazi and 798 of Sephardic Jews. We have also studied the impact of selected new genetic variants on POR activity and identified fourteen amino acid variations, two of which we have studied in detail to establish their influence on POR activity. Using the available human POR three-dimensional structure, we then modelled the newly identified variants...

FundaÃÃo de Amparo à Pesquisa do Estado do Cearà / A caracterizaÃÃo do fenÃtipo acetilador e oxidativo das atividades das enzimas metabolizadoras à de fundamental relevÃncia para avaliaÃÃo da resposta farmacolÃgica aos vÃrios agentes terapÃuticos. Os estudos de fenotipagem e genotipagem tÃm como finalidade detectar e mensurar polimorfismos nestas enzimas. O estudo teve como objetivo determinar o perfil fenotÃpico da atividade metabolizadora das enzimas CYP1A2, CYP2A6, XO e NAT2 em uma amostra de oitenta voluntÃrios saudÃveis da populaÃÃo do estado do CearÃ, utilizando a cafeÃna como fÃrmaco âprobeâ. Os voluntÃrios foram avaliados atravÃs de consulta mÃdica para confirmaÃÃo do estado de higidez; as funÃÃes metabÃlicas, hepÃtica e renal foram avaliadas atravÃs de exames laboratoriais hematolÃgicos e bioquÃmicos. O protocolo clÃnico consistiu de um internamento onde os voluntÃrios receberam um comprimido de 200mg de cafeÃna e coletas de sangue e urina. As amostras de sangue seguiram os seguintes intervalos a partir da administraÃÃo: 0; 0:05; 0:15; 0:25; 0:35; 0:45; 1:00; 1:15; 1:30; 2; 4; 6; 8; 12 e 24 horas. A urina foi colhida em seu volume total durante as 10 horas apÃs a administraÃÃo, o volume urinÃrio total foi quantificado e uma alÃquota foi armazenada para determinaÃÃo dos metabÃlitos. O protocolo analÃtico consistiu de dosagem por HPLC das concentraÃÃes plasmÃticas de cafeÃna e das concentraÃÃes urinÃrias dos cinco principais metabÃlitos da cafeÃna: 1U â Ãcido 1-metilÃrico; 17U â Ãcido 1,7-dimetilÃrico; 1X â metilxantina; 17X â 1,7-dimetilxantina e AFMU â 5-acetilamino-6-formilamino-3-metiluracil. A partir das concentraÃÃes molares urinÃrias dos metabÃlitos foram determinadas as atividades das enzimas: CYP1A2 = (AFMU + 1X + 1U)/ 17U; CYP2A6 = 17U / (AFMU + 1X + 1U + 17X + 17U); NAT2 = AFMU/ (AFMU + 1X + 1U) e XO = 1U/ (1X + 1U). Os valores das atividades enzimÃticas foram submetidos a anÃlise por estatÃstica descritiva (histograma) nÃo apresentando distribuiÃÃo normal. Foram identificados pelo menos trÃs fenÃtipos metabolizadores para as enzimas CYP1A2, NAT2 e XO. Com base na atividade de cada enzima os voluntÃrios foram divididos em dez grupos organizados categoricamente. Os voluntÃrios dos grupos de maior e menor atividade enzimÃtica foram escolhidos para determinaÃÃo dos parÃmetros farmacocinÃticos (ASC; t1/2; ke; Vd/Kg; CL). A comparaÃÃo destes parÃmetros (teste âtâ de Student) entre os dois grupos apresentou diferenÃas significantes (p<0,05). Nossos resultados sugerem a existÃncia de polimorfismo nas enzimas estudadas de acordo com a heterogeneidade das atividades enzimÃticas na amostra da populaÃÃo de voluntÃrios saudÃveis do estado do CearÃ. / Characterization of acetylator and oxidative metabolizers phenotypes is of clinical relevance, as it has been shown that the pharmacological response of several therapeutic agents. The studies of phenotypage and genotipage have as purpose to detect and to mensurar polimorfismos in these enzymes. In this study, we assessed in vivo activities of CYP1A2, CYP2A6, Xantine Oxidase (XO) and NAT2 in sample of 82 volunteers (40 men and 42 women) from Cearà â Brazil using caffeine as probe. The volunteers were free from significant cardiac, hepatic, renal, pulmonary, gastrointestinal and hematological disease, as assessed by physical investigation, ECG and hematological and biochemical tests. The study was open, the volunteers were hospitalized, having already had a normal evening meal, and after an overnight fast they received a single 200 mg dose of caffeine tablet. Blood samples were collected in regular interview ( 0; 5min; 15min; 25min; 35min; 45min; 1h; 1,25h; 1,5h; 2h; 4h; 6h; 8h; 12h and 24 hour) after the administration of caffeine. Urine was harvested in its total volume during the 10 hours after the administration of caffeine, the total urinÃrio volume was quantified and an aliquot one was stored for determination of the metabÃlitos. The caffeine was determined in human plasma and the metabolites were determined in human urinary by HPLC using teobromina as an internal standard. The metabolites assessed were: 1U â 1-methyluric acid; 17U â 1, 7-dimethyluric acid; 1X â 1-methylxantine; 17X â 1, 7-1-dimethylxantine e AFMU â 5-acetylamino-6-formylamino-3methyluracil. The following molar ratios were calculated as an index for CYP1A2 activity [(AFMU + 1X + 1U)/ 17U]; CYP2A6 activity [17U / (AFMU + 1X + 1U + 17X + 17U)]; NAT2 activity [AFMU/ (AFMU + 1X + 1U)] and Xanthine Oxidase activity [1U/ (1X + 1U)]. The enzymatic activities were plotted in histogram. The frequency distributions of the CYP1A2, CYP2A6, XO and NAT2 were not normally distributed, showing three phenotypes. Afterwards the volunteers were classified by enzymatic activities in ten groups, and the lowest and highest metabolizers groups were chosen to determine the following pharmacokinetic parameters: AUC0-24, t1/2, ke, CL and Vd/Kg. The comparison of these parameters (test âtâ de Student) between these two groups it showed significant differences (p<0.05). Our findings may suggest that the heterogeneous population assessed (health volunteers from Cearà - Brazil) has shown polymorphism in the enzymatic activities of the CYP1A2, NAT2, XO and CYP2A6

Alterações morfológicas no tecido ósseo têm sido descritas nos usuários de hipoglicemiantes orais da classe das tiazolidinedionas (TZDs). Hipotetiza-se que alguns genes relacionados com a osteogênese e osteoclastogênese podem ser influenciados pelo tratamento farmacológico, entretanto, o exato mecanismo ainda não está bem esclarecido. O objetivo do estudo foi avaliar o efeito da pioglitazona no remodelamento ósseo através de genes envolvidos na osteoclastogênese em indivíduos recentemente diagnosticados com DM2 e modelos animais, com a finalidade de identificar marcadores genéticos sensíveis de alterações ósseas. Foram convidados para participar do estudo 199 indivíduos (100 diabéticos e 99 normoglicêmicos), no ambulatório de dislipidemias do Instituto Dante Pazzanese de Cardiologia. Os indivíduos diabéticos foram tratados com pioglitazona (15, 30, 45, 45 mg/ dia/ via oral) por 16 semanas. Foram colhidas amostras de sangue, antes e após o tratamento para avaliações laboratoriais, extração de DNA genômico e de RNA total. Os polimorfismos e a expressão do mRNA nas células sanguíneas foram determinados pela PCR em tempo real através do sistema TaqMan®. Para o estudo em modelo animal após a indução da dieta hiperlipídica por 32 semanas, foram utilizados 12 camundongos machos da linhagem C57BL/J6, os quais foram divididos em três grupos: controle (n=4); diabéticos induzidos pela dieta hiperlipídica (DH, n=4) e diabéticos induzidos pela dieta hiperlipídica e tratados com pioglitazona 35mg/Kg/dia por 16 semanas (DHP, n=4). Para os grupos experimentais foram colhidos: amostras de sangue, para exames laboratoriais; fêmures, para a extração do RNA total; e tíbias, para determinação dos parâmetros histomorfométricos. Os pacientes DM2 apresentaram diminuição nas concentrações séricas de osteocalcina e na expressão de OPG e aumento na expressão de VDR em comparação ao grupo NG (p<0,05). A expressão de RANKL e IL6 foi maior entre as mulheres, enquanto que a expressão de PPARG foi maior entre os homens com DM2 em comparação ao grupo NG (p=0,032). Pacientes DM2 antes do tratamento apresentaram glicemia e expressão do mRNA de IL6 negativamente associados ao cálcio ionizado, enquanto que as transcrições de TNFA e VDR foram associadas positivamente e negativamente com bALP respectivamente (p<0,05). O tratamento com pioglitazona reduziu a glicemia de jejum, glicemia pós-prandial, insulina, HOMA-IR, triglicerídeos, VLDL-C, tALP e bALP e aumentou a HDL, tACP, TNF-&#945; e a transcrição de OPG (p<0,05). A glicemia basal associou-se positivamente com o cálcio ionizado. A expressão basal de OPG foi associado negativamente com tALP, enquanto que a expressão basal de TNFA foi associada positivamente com tALP e negativamente com tACP. A expressão basal IL6 foi associada positivamente com tALP, enquanto que a expressão basal de VDR foi associada negativamente com osteocalcina e positivamente com bALP em resposta ao tratamento (p<0,05). O polimorfismo RANK rs1805034 foi associado com redução na transcrição do gene RANK nos indivíduos DM2 e com o remodelamento ósseo após o tratamento com pioglitazona (p<0,05). O polimorfismo RANKL rs9525641 foi associado com aumento da transcrição gênica de RANKL nos indivíduos NG e DM2 e melhora da resposta farmacológica nos indivíduos DM2 tratados com pioglitazona (p<0,05). O polimorfismo rs3102735 do gene OPG foi associado com aumento da formação óssea nos indivíduos DM2 antes e após o tratamento (p<0,05). O genótipo CG do polimorfismo OPG rs2073618 foi associado com alteração da transcrição de OPG no grupo DM2 pré e pós-tratamento (p<0,05). O polimorfismo PPARG rs1801282 foi associado com menor risco para o desenvolvimento de diabetes (p<0,05). O polimorfismo PPARG rs2972162 foi associado com melhora da resistência insulínica nos indivíduos DM2 tratados com pioglitazona (p=0,017). O polimorfismo ESRI rs9340799 foi associado com redução da formação óssea nos indivíduos DM2 (p=0,038). Nos camundongos, após a indução da dieta hiperlipídica por 32 semanas, observou-se aumento do peso, da glicemia, do colesterol total, da expressão do mRNA de RANK, RANKL, IL6 e TNFA em fêmures e aumento de Tb.Sp e diminuição de BV/TV em comparação ao grupo controle (p<0,05). O tratamento com pioglitazona diminuiu a expressão de TNFA (p=0,028). As medidas histomorfométricas não alteraram-se após o tratamento (p>0,05). Os resultados sugerem que o estado hiperglicêmico e o tratamento influenciam os marcadores bioquímicos e moleculares. Os polimorfismos dos genes RANK, RANKL, OPG e ESRI parecem estar envolvidos no remodelamento ósseo independentemente da hiperglicemia e do tratamento e os polimorfismos do gene PPARG parecem estar envolvidos com menor risco para desenvolver diabetes e com a melhora da resistência insulínica em resposta ao tratamento com pioglitazona. / Morphological changes in bone tissue have been reported in users of oral hypoglycemic class of thiazolidinediones (TZDs). It is hypothesized that some genes related to osteogenesis and osteoclastogenesis may be influenced by pharmacological treatment, however, was not aware exact mechanism. The study aims was to evaluate pioglitazone effect on bone remodeling through genes involved in osteoclastogenesis in individuals newly diagnosed with DM2 and animal models, in order to identify sensibles genetics markers of bone alterations. Were invited to participate in study 199 patients (100 diabetics and 99 normoglycemic), in dyslipidemia ambulatory of Institute Dante Pazzanese of Cardiology. Diabetic subjects were treated with pioglitazone (15, 30, 45 or 45 mg /day/oral) for 16 weeks. Blood samples were collected before and after treatment for laboratory evaluations, extraction of genomic DNA and total RNA. Polymorphisms and mRNA expression in blood cells was determined by real time PCR using TaqMan® system. For study in animal model after 32 weeks of fat diet induction, was used 12 male mice C57BL/J6, which were divided into three groups: control (n=4); induced diabetic fat diet (DH, n=4) and induced diabetic fat diet and treated with pioglitazone 35mg/Kg/day for 16 weeks (DHP, n=4). For experimental groups were collected: blood samples for laboratory tests; femurs, for extraction of total RNA; and tibias, to determine histomorphometric parameters. DM2 patients showed decrease in serum osteocalcin and OPG expression and increased VDR expression compared to NG group (p<0.05). RANKL and IL6 expression were higher among women, whereas PPARG expression was higher among men with DM2 compared to NG group (p=0,032). DM2 patients before treatment showed blood glucose and IL6 mRNA expression negatively associated with ionized calcium, whereas TNFA and VDR transcription are positively and negatively associated with bALP respectively (p<0.05). Pioglitazone treatment reduced fasting glucose, postprandial glucose, insulin, HOMA-IR, triglycerides, VLDL-C, tALP and bALP and increased HDL, tACP, TNF-&#945; and OPG transcription (p<0.05). Basal blood glucose was positively associated with ionized calcium. Basal OPG expression was negatively associated with tALP, whereas basal TNFA expression was positively associated with tALP and negatively with tACP. Basal IL6 expression was positively associated with tALP, whereas basal VDR expression was negatively associated with osteocalcin and positively with bALP in response to treatment (p<0.05). RANK rs1805034 polymorphism was associated with RANK gene transcription reduction in subjects with DM2 and bone remodeling after treatment with pioglitazone (p<0.05). RANKL rs9525641 polymorphism was associated with increased RANKL gene transcription in NG and DM2 subjects and pharmacological response improvement in DM2 subjects treated with pioglitazone (p<0.05). OPG rs3102735polymorphism was associated with increased bone formation in DM2 subjects before and after treatment (p<0.05). CG genotype of OPG rs2073618 polymorphism was associated with OPG transcription change in DM2 group before and after treatment (p<0.05). PPARG rs1801282 polymorphism was associated with lower risk for diabetes development (p<0.05). PPARG rs2972162 polymorphism was associated with insulin resistance improvement in DM2 subjects treated with pioglitazone (p=0,017). ESRI rs9340799 polymorphism was associated with reduced bone formation in DM2 subjects (p=0,038). In mice, after 32 weeks of fat diet induction, was observed increase weight, blood glucose, total cholesterol and RANK, RANKL, IL6 and TNFA mRNA expression in femurs and Tb.Sp increase and BV/TV decrease compared to control group (p<0.05). Treatment with pioglitazone decrease TNFA (p=0,028). Histomorphometrics measurements not change after treatment (p>0.05). Results suggest that hyperglycemic state and treatment influence biochemical and molecular markers. RANK, RANKL, OPG and ESRI polymorphisms seens to be involved in bone remodeling regardless of hyperglycemia and treatment and PPARG gene polymorphisms seens to be associated with lower risk for diabetes development and with insulin resistance improvement in response to treatment with pioglitazone.

No description available.