A enzima glutationa peroxidase (GPx-1) possui um importante papel na detoxificação de espécies reativas de oxigênio. Neste estudo foi verificada a influência do polimorfismo Pro198Leu da GPx-1 nos níveis de malondialdeído (MDA) e proteínas carboniladas de 84 voluntários expostos ambientalmente ao chumbo (28 homens e 56mulheres; idade: 18 a 60 anos). Os genótipos para o polimorfismo Pro198Leu da GPx-1 foram determinados por PCR seguido por digestão com enzima de restrição. Chumbo no plasma (Pb_P) e chumbo no sangue total (Pb_S) foram determinados por espectrometria de massas com plasma indutivamente acoplado e espectrometria de absorção atômica com forno de grafite, respectivamente. As freqüências alélicas para os alelos T (que codifica o aminoácido prolina) e C (que codifica o aminoácido leucina) da GPx-1 foram 0,73 e 0,27, respectivamente. Os voluntários foram separados em dois grupos, de acordo com a presença ou ausência do alelo que codifica leucina. Não foi encontrada diferença significativa para as concentrações de Pb_P e Pb_S, bem como para os marcadores de estresse oxidativo entre os grupos. Deste modo, os resultados obtidos sugerem que o polimorfismo Pro198Leu da GPx-1 não torna indivíduos ambientalmente expostos ao chumbo, mais suscetíveis aos danos do estresse oxidativo induzidos pelo metal. / Glutathione peroxidase (GPx-1) plays an important role in scavenger reactive oxygen species (ROS). This study was carried out to assess the effects of GPx-1 gene polymorphism (Pro198Leu) on malondialdehyde (MDA) and carbonyl protein ratio in 84 subjects environmentally exposed to lead. Genotypes for the GPx-1 Pro198Leu polymorphism were determined by PCR and restriction fragment length digestion. Lead in plasma (Pb_P) and lead in total blood (Pb_B) were determined by inductively coupled plasma mass spectrometry and by graphite furnace atomic absorption spectrometry, respectively. The allele frequencies for T (which express the proline aminoacid) and C (which express the leucine aminoacid) were 0.73 e 0.27, respectively. The volunteers were divided in two groups, according to the presence or absence of C allele. No significant differences were found in Pb_P and Pb_B, as well as for the other oxidative biomarkers between the groups. Therefore, the obtained results suggest that the GPx-1 Pro198Leu polymorphism do not affect individuals environmentally exposed to lead greater susceptible to oxidative damage induced for this metal.

O chumbo (Pb) é um metal altamente tóxico no qual os sinais de intoxicação variam bastante ao considerar as diferenças interindividuais. Um dos principais mecanismos de toxicidade do Pb ocorre pela inibição da enzima ácido delta aminolevulínico desidratase (ALAD) no sistema hematopoiético. O Pb também desempenha um importante papel no desbalanço do estado redox, pois sabe-se que ele tem o potencial de aumentar a concentração de espécies reativas de oxigênio (EROS) e inibir enzimas antioxidantes, como por exemplo a glutationa peroxidase (GPx). No entanto, poucos estudos avaliaram estes parâmetros em população ocupacionalmente exposta brasileira. Assim, o presente estudo, objetiva estudar a correlação entre as concentrações de Pb no sangue (Pb-S) de trabalhadores de fábricas de bateria e as atividades das enzimas ALAD e GPx associados com os polimorfismos genéticos da ALAD e GPx. Para tal, foram utilizadas 278 amostras de sangue de trabalhadores expostos ao Pb. As determinações de Pb foram realizadas por espectrometria de massas com plasma acoplado indutivamente (ELAN DRCII Perkin- Elmer). As genotipagens dos polimorfismos genéticos da ALAD e da GPx foram realizadas pela Reação em Cadeia da Polimerase (PCR) em tempo real e as atividades das enzimas ALAD e GPx foram determinadas no sangue por espectrofotometria de UV/VIS. A média da concentração de Pb-S foi de 22,8 ± 14,7 ?g/dL. Foram observadas correlações negativas entre Pb-S e atividade da ALAD (rs -0,24 p<0,01) e Pb-S e atividade de GPx (rs -0,27 p<0,05). Também foi verificada correlação negativa entre a porcentagem de inibição da ALAD e a atividade de GPx (rs -0,21 p<0,01). Em relação aos polimorfismos genéticos, não observamos associação entre os genótipos do ALAD (? -0,19 P>0,05) e GPx (genótipo CT: ? -1,37 P>0,05; genótipo TT: ? -8,37 P>0,05) e a concentração de Pb-S. Não foram observadas associações entre o polimorfismo rs1800668 e a atividade de GPx (genótipo CT: ? -0,016 P>0,05; genótipo TT: ? -0,004 P>0,05). No entanto, foi constatada uma associação entre o genótipo ALAD 1-1 do polimorfismo rs1800435 e a atividade da enzima ALAD (? 3,5 P<0,05). Neste sentido, os resultados deste estudo mostram que o genótipo ALAD 1-1 do polimorfismo rs1800435 do gene ALAD está associado a uma maior atividade da enzima ALAD nos indivíduos expostos ao Pb. Além disso, o polimorfismo rs1800668, localizado no gene que codifica a enzima GPx, não modula a atividade desta enzima nos indivíduos expostos ao metal. Ambos os polimorfismos dos genes ALAD e GPx parecem não influenciar nas concentrações de Pb-S na população estudada. / Lead (Pb) is a highly toxic metal which signs of intoxication vary greatly when considering the interindividual differences. One of the main mechanism of toxicity of Pb is due to inhibition of the enzyme acid delta aminolevulinic dehydratase (ALAD) in the hematopoietic system. Pb also plays an important role in the redox state of unbalance, since it is known that it has the potential of increasing the concentrations of reactive oxygen species (ROS) and inhibit antioxidant enzymes such as glutathione peroxidase (GPx). However, few studies have evaluated these parameters in Brazilian population occupationally exposed. Thus, the present study aims to study the correlation between the concentrations of Pb in the blood (B-Pb) of workers of battery factories and the activities of ALAD and GPx enzymes associated with genetic polymorphisms in ALAD and GPx. Then, blood samples of 278 workers exposed to Pb were collected. Pb was measured by Inductively Coupled Plasma-Mass Spectrometry (Perkin-Elmer ELAN DRCII). The genotyping of genetic polymorphisms of ALAD and GPx was performed by Real-Time Polymerase Chain Reaction (PCR) and activity of ALAD and GPx enzymes in the blood was determined by spectrophotometry UV / VIS. The mean concentration of B-Pb was 22.81 ± 14.73 mg / dL. Negative correlation was found between B-Pb and ALAD activity (rs -0.24 p < 0.01) and B-Pb and GPx activity (rs -0.27 p < 0.05). There was also a negative correlation between the percentage of inhibition of ALAD and GPx activity (rs -0.21 p < 0.01). Considering the genetic polymorphisms, no association between ALAD genotypes (? -0.19 P < 0.05) and GPx (CT genotype: ? -1.37 P > 0.05, TT genotype : ? -8.37 P > 0.05 ) and the concentration of B-Pb. No association between rs1800668 polymorphism and GPx activity (CT genotype: ? -0.016 P > 0.05, TT genotype : ? -0.004 P > 0.05). However an association was found between ALAD 1-1 rs1800435 genotype and ALAD enzyme activity (? 3.5 P < 0.05). Accordingly, the results of this study show that the ALAD 1-1 genotype of rs1800435 polymorphism in ALAD gene is associated with increased activity of ALAD enzyme in individuals exposed to Pb. Furthermore the rs1800668 polymorphism located in a gene encoding the enzyme GPx does not modulate the activity of this enzyme in individuals exposed to the metal. Both polymorphisms of ALAD and GPx genes seem to have no influence in the concentrations of B-Pb in the population studied.

O selênio (Se), mineral traço essencial para o ser humano, exerce sua função no organismo por meio de sua participação nas selenoproteínas, dentre as quais destacam-se as glutationas peroxidase (GPx), importantes no sistema de defesa antioxidante. São conhecidas sete isoformas da GPx, sendo a GPx 1 (citosólica) a mais abundante. Como a atividade dessa enzima encontra-se fortemente correlacionada com a concentração sanguínea de Se, este parâmetro é normalmente utilizado como indicador do balanço metabólico do mineral. A concentração de Se nos alimentos depende do local onde foram cultivados e reflete diretamente o teor do mineral no solo, sendo os alimentos da região Norte e Nordeste mais ricos nesse nutriente quando comparados às demais regiões brasileiras. O consumo inadequado de Se provoca uma diminuição da atividade dessa enzima, o que por sua vez pode afetar a proteção antioxidante. Além disso, estudos vêm mostrando que o polimorfismo Pro198Leu no gene da GPx1 também pode diminuir a atividade da GPx. O presente estudo se propôs a verificar a relação entre esse polimorfismo e o estado nutricional relativo ao selênio de uma população adulta residente no município de Fortaleza/Ceará. A população do estudo foi constituída por 176 indivíduos (79 homens e 97 mulheres), com média de idade de 30,4 ± 8,9 anos. Foram realizadas a avaliação da composição corporal por antropometria e a avaliação do consumo alimentar, por meio de três recordatórios de 24h. Os biomarcadores para avaliação do estado nutricional dos indivíduos quanto ao selênio foram: a concentração de Se plasmático e eritrocitário e a atividade da GPx total no eritrócito. Também foi determinado o polimorfismo Pro198Leu no gene que codifica para a GPx 1 por meio de PCR em Tempo Real. Foram encontradas concentrações médias de 62,6 &#181;g/L para Se plasmático e de 101,5 &#181;g/L para Se eritrocitário, não sendo observada correlação entre os dois. A média de ingestão de selênio foi de 76,88 &#181;g/dia, apresentando correlação positiva com o consumo de energia e proteína. Separando por gênero, encontrou-se que as médias de Se plasmático e de Se ingerido dos homens foram significativamente maiores que as das mulheres (p<0,05). A média da atividade da GPx foi de 38,6 U/g Hb. Para o polimorfismo Pro198Leu no gene que codifica para a GPx 1, foram encontrados 96 (54,5%) indivíduos com genótipo selvagem Pro/Pro, 67 (38,1%) indivíduos Pro/Leu e 13 (7,4%) indivíduos Leu/Leu. A frequência encontrada estava em equilíbrio de Hardy-Weinberg. Não foram encontradas diferenças estatísticas significativas nas concentrações de selênio entre os grupos de acordo com o genótipo, no entanto, para a atividade da GPx, houve diferença entre o grupo Pro/Pro e Pro/Leu (p<0,05). No grupo Pro/Pro, foi encontrada correlação positiva entre a concentração de selênio eritrocitário e a atividade eritrocitária total da GPx (p<0,05). Desta forma, podemos concluir que o estado nutricional da população estudada estava adequado com relação ao selênio e que o polimorfismo Pro198Leu apresentou influência sobre a atividade da GPx, que apresentou-se reduzida nos indivíduos que apresentaram alelo variante em seu genótipo. / Selenium (Se) is an essential trace mineral for humans which exerts its function in the body in selenoproteins, among which we highlight the glutathione peroxidase (GPx), important in the antioxidant defense system. There are seven known GPx isoforms and the GPx 1 (cytosolic) is the most abundant. As the activity of this enzyme is strongly correlated with selenium blood concentration this parameter is usually used as an indicator of the mineral metabolic balance. Selenium concentration in food depends on the location where they were grown and directly reflects the soil mineral content. Food in the North and Northeast are richer in selenium when compared to other Brazilian regions. Inadequate consumption of Se causes a decrease in GPx activity, what affects the antioxidant protection. Furthermore, studies have shown that the Pro198Leu polymorphism at GPx1 gene of can also decrease the activity of GPx. Our study aimed to investigate the relationship between this polymorphism and nutritional selenium status in an adult population living in Fortaleza/Ceará/Brazil. The study population consisted of 176 individuals (79 men and 97 women) with a mean age of 30.4 ± 8.9 years. The body composition was assessed by anthropometry and the food intake assessment was done using three 24-hour records. The selenium Se concentration was measured in plasma and erythrocyte and GPx activity was measured in erythrocytes. The Pro198Leu polymorphism at GPx 1 gene was determined by Real Time PCR. We found concentrations of 62.6 &#181;g/L for Se plasma and 101.5 &#181;g/L for erythrocyte. There was no correlation observed between these two markers. The average selenium intake was 76.88 &#181;g/day and it showed positively related to the energy and protein consumption. Separating by gender, men selenium plasma concentration and selenium consumption were significantly higher than for women (p < 0.05). The GPx activity was 38.6 U/g Hb. The Pro198Leu polymorphism at GPx 1 gene frequency were 96 (54.5%) subjects with Pro/Pro, 67 (38.1%) subjects Pro/Leu and 13 (7.4%) individuals Leu/Leu. The rate found was in Hardy -Weinberg equilibrium. There were no statistically significant differences in selenium concentrations between groups according to the genotype. However, for GPx activity we found difference between the group Pro/Pro and Pro/Leu (p< 0.05). We also found positive correlation between Se erythrocyte concentration and GPx activity in Pro/Pro group (p<0.05). Thus, we conclude that the selenium nutritional status of the population studied was adequate and that Pro198Leu polymorphism showed influence on the activity of GPx, which was lower in individuals with the variant allele in their genotype.

O selênio (Se) é um mineral essencial para o corpo humano, com ação no sistema imunológico, na glândula tireoide, dentre outras funções. É um nutriente importante na proteção das células contra os efeitos dos radicais livres, já que faz parte da enzima glutationa peroxidase (GPX). O consumo alimentar de Se é dependente da concentração desse elemento nos alimentos que varia em função da concentração do mesmo nos solos. Deste modo, este estudo teve como objetivo geral avaliar o estado nutricional relativo ao selênio de indivíduos adultos da cidade de Manaus-AM, por ser considerada região com solo rico nesse nutriente. Casuística: o estudo foi do tipo transversal baseado em amostra não probabilística de conveniência. A população estudada foi composta por 124 voluntários de ambos os gêneros com idade entre 20 a 59 anos. Critérios de inclusão: não ser atleta de elite, não ser etilista, não apresentar doenças crônicas não transmissíveis e não fazer o uso de suplementos vitamínico-minerais e/ou anti-inflamatórios. Não foram incluídos no estudo os participantes que não entregaram os formulários e questionários solicitados (alimentação e/ou ficha cadastral) e/ou que não compareceram para coleta de sangue. A coleta de sangue foi em jejum de 8 a 12 horas para determinação de selênio no plasma e eritrócitos e da atividade da GPX nos eritrócitos, também foram avaliados o consumo alimentar e as medidas antropométricas. Os resultados antropométricos mostraram que 60,0% dos participantes apresentava excesso de peso, a distribuição dos macronutrientes ficou dentro do recomendado pelas DRIs (Dietary Reference Intakes), a ingestão média de selênio ficou acima do recomendado (72±48,0&#181;g/dia), a concentração média para o selênio plasmático foi de 111,4± 37,0 &#181;g/L, de selênio eritrocitário 211± 62,4 &#181;g/L e a atividade da GPX foi de 73± 21,9 U/g Hb, não sendo observada correlação entre as concentrações obtidas para plasma e eritrócito, porém com correlação positiva para Se eritrocitário e atividade da GPX (r= 0,393). Pode-se, portanto concluir do presente estudo que a boa parte dos indivíduos avaliados, habitantes da cidade de Manaus, foi considerada deficiente, apesar de consumirem quantidade desse mineral consideradas adequadas porém, nenhum apresentando parâmetros bioquímicos de deficiência, a maioria dentro da normalidade, entretanto, com um percentual de indivíduos apresentando risco de toxicidade. / Selenium (Se) is an essential mineral for the human body, with function on the immune system, thyroid glands and other ones. It is an important nutrient in protecting cells against the effects of free radicals because it is part of glutathione peroxidase enzyme (GPX). Selenium intake depends on the mineral concentration in aliments which varies based on the mineral content in soils. Thus, this study had as main objective to evaluate the nutritional status related to selenium on adults in Manaus-AM because it is considered as a region of high selenium soil content region. Casuistic: This study was transversal type based in a non-probabilistic convenience sample. The population consisted of 124 volunteers (both genders) aged from 20 to 59 years. Inclusion criteria: To not be an elite athlete or alcoholic, do not have chronic non-communicable diseases and do not use vitamin-mineral supplements and/or anti-inflammatory pills. People who did not submit the requested forms and questionnaires (registration form and/or alimentation) and/or did not attend for blood collection were not included. Blood collection was fasting from 8 to 12 hours for determining selenium in plasma and erythrocytes and the activity of GPX in erythrocytes. Food intake and anthropometric measures were also evaluated. Anthropometric results showed that 60,0% were overweight, macronutrient distribution was within the recommended by DRIs (Dietary Reference Intakes). Average selenium intake was above recommended (72±48,0 &#181;g/day), mean plasmatic selenium concentration was 111,4 ± 37,0 &#181;g/L, for erythrocytes selenium was 211 ± 62,4 &#181;g/L and activity of GPX average was 73 ± 21,9 U/g Hb. No correlation between concentration of Se in plasma and erythrocyte, however, there was positive correlation between GPX activity and Se concentration in erythrocytes (r=0,393). Finally, we conclude that most of the people that were evaluated, citizens of Manaus city, were considered as deficient, although they consumed adequate quantities of this mineral. However, none of them showed biochemical deficiency parameters, most within normality, but with a percentage of them showing toxicity risk.

Dans le but de protéger la levure Saccharomyces cerevisiae pendant la déshydratation, deux stratégies ont été envisagées pour améliorer la survie cellulaire. D’une part, le renforcement de la résistance à l’oxydation (protection interne) a été réalisé par l’enrichissement en glutathion intracellulaire de la levure. D’autre part, l’encapsulation par la méthode couche-par-couche (layer-by-layer) en utilisant deux biopolymères β-lactoglobuline et alginate a été utilisée pour la protection externe de la levure. Un milieu adéquat ainsi que des conditions favorables de culture permettant l’enrichissement en glutathion intracellulaire ont été déterminés. La composition du milieu est riche en nutriments et précurseurs du glutathion se composant de 30 g/L de glucose, 30 g/L d’extrait de levure, 0,6 g/L de KH2PO4 et 0,6 g/L de cystéine. Le succès de l’encapsulation a été confirmé par différentes méthodes d’analyse comme la zétamétrie, la microscopie électronique et la spectroscopie infrarouge. Le dépôt des trois couches de β-lactoglobuline/alginate/β-lactoglobuline n’a pas affecté l’intégrité membranaire et la croissance de la levure. De plus, la perméabilité membranaire n’a pas été empêchée par la présence de cette barrière biopolymérique. L’effet de l’enrichissement en glutathion et de l’encapsulation sur la résistance de la levure dans les conditions de déshydratation choisies a été mesuré par l’estimation de la survie, les modifications biochimiques et le métabolisme des cellules. Les résultats ont montré que la levure enrichie en glutathion présente une meilleure survie après la déshydratation. De plus, la survie de la levure est améliorée par l’encapsulation, en particulier pendant la déshydratation à 45 °C. L’analyse des résultats d’infrarouge a relevé un point commun dans la relation entre les propriétés biochimiques de la cellule et la survie cellulaire. Plus le groupement méthyle (CH3) est important, plus la survie est importante. Le rôle du glutathion dans la protection de la levure a également été mis en évidence pendant la déshydratation. Il semblerait que l’action du glutathion oxydé ait été mise en place après l’intervention du glutathion réduit. De plus, la protection interne par le glutathion a permis à la levure de résister au stress chimique alors que la protection externe par encapsulation renforce la résistance contre les stress physique ou mécanique. Les résultats de la thèse pourront être utiles pour la recherche des nouvelles technologies de production et de protection des levures fragiles actives. / In order to protect the Saccharomyces cerevisiae yeast during dehydration, two strategies were used to improve cell survival. Strengthening cell resistance (internal protection) was performed by improving the concentration of intracellular glutathione in yeast. Encapsulation was performed by layer-by-layer method using two biopolymers β-lactoglobulin and alginate for the external protection of the yeast. The adequate medium culture and favorable growing conditions for glutathione enhancement were determined. The composition of the culture medium is rich in nutrients and glutathione precursors consisting in 30 g/L glucose, 30 g/L yeast extract, 0.6 g/L KH2PO4 and 0.6 g/L cysteine. The success of encapsulation was confirmed by different analytical methods such as zetametry, electron microscopy and infrared spectroscopy. The deposition of three layers of β-lactoglobulin/alginate/β-lactoglobulin did not affect membrane integrity and the growth of yeast. Furthermore, the membrane permeability was not affected by the presence of this biopolymer barrier. The effect of glutathione and encapsulation on the resistance of the yeast in dehydrating conditions was measured by the estimation of cell survival, biochemical modification and cellular metabolism. The results showed that the glutathione-enriched yeast present higher survival after dehydration. Moreover, the survival of the yeast was improved by the encapsulation. These observations were clearly obtained in dehydration conditions at 45 °C. Infrared analysis identified a common point in the relationship between biochemical property and cell survival. Higher survival was observed when the yeast was characterized by methyl group (CH3). The role of glutathione in yeast protection was highlighted during dehydration. It seemed that the action of oxidized glutathione was set up after the intervention of reduced glutathione. In addition, the internal protection by glutathione allowed the yeast to resist to chemical stress while the external protection by encapsulation enhances the resistance against physical or mechanical stress. These results can be useful for the research of new technology in fragile yeast production and protection.

Abstract Reactive metabolites are believed to be responsible for rare but serious idiosyncratic adverse drug reactions (IADRs) that have led to the withdrawal of numerous drugs from the market. This has resulted in major harm to patients, economic losses for the pharmaceutical companies and represents a serious problem in drug development. Reactive metabolites can be studied by trapping them with suitable nucleophiles, most commonly with glutathione. The glutathione conjugates formed in these reactions can be analyzed with liquid chromatography mass spectrometry (LC/MS) techniques. In this study, new in vitro methods for the detection and analysis of reactive metabolites were developed. The suitability for reactive metabolite screening of different enzyme sources commonly used in vitro were compared. It was found that sub-cellular fractions yielded significantly larger amounts of glutathione-trapped reactive metabolites as compared to the amounts obtained from intact hepatocytes. Additionally, different metabolites were detected in some cases. Biomimetic metalloporphyrin catalysts were tested for their ability to produce larger amounts of glutathione-trapped metabolites relative to liver S9 fraction incubations. An increase in reactive metabolite production was observed with biomimetic models, but not all of the metabolites produced by liver S9 were observed. The glutathione conjugates of pulegone and of its metabolite menthofuran were analyzed with LC/MS/MS, and the fragmentation spectra of N- and S-/N- di-linked glutathione conjugate were interpreted in detail for the first time. These results will enable more efficient screening of reactive metabolites of furan-containing compounds. Acyl glucuronides are metabolites produced from carboxylic acid-containing compounds and can be reactive. A good correlation was found between the acyl migration half-life and the tendency of a drug to cause IADRs. The carboxylic moiety can also be metabolized to yield acyl coenzyme A (CoA) conjugates that may be more reactive than their corresponding acyl glucuronides. The formation of CoA conjugates and additional conjugates formed from them was found to be more likely with drugs that cause IADRs. / Tiivistelmä Reaktiivisten metaboliittien uskotaan olevan syypää tietyntyyppisiin harvinaisiin, mutta vakaviin idiosynkraattisiin lääkehaittavaikutuksiin, jotka ovat johtaneet useiden lääkeaineiden poistamiseen markkinoilta. Ne ovat aiheuttaneet merkittäviä haittoja potilaille, tappioita lääkeyhtiöille ja ovat vakava ongelma lääkekehityksessä. Reaktiivisia metaboliitteja voidaan tutkia vangitsemalla niitä sopivilla nukleofiileillä, yleisimmin glutationilla. Muodostuneet glutationikonjugaatit voidaan sitten analysoida nestekromatografia / massaspektrometrisin tekniikoin. Tässä tutkimuksessa kehitettiin uusia in vitro tapoja havaita ja analysoida reaktiivisia metaboliitteja. Tavallisimmin käytettyjen entsyymilähteiden soveltuvuutta testattiin reaktiivisten metaboliittien seulontaan. Solufraktioiden havaittiin tuottavan huomattavasti suurempia määriä glutationi-vangittuja reaktiivisia metaboliitteja kuin elävät solut. Lisäksi eri metaboliitteja havaittiin joillekin aineille eri entsyymilähteissä. Biomimeettisen metalliporfyriinikatalyytin kykyä tuottaa suurempia määriä glutationilla vangittuja reaktiivisia metaboliitteja testattiin vertaamalla sitä maksan S9 fraktioon. Vaikka katalyytillä pystyi tuottamaan suurempia määriä reaktiivisia metaboliitteja, kaikkia S9 fraktiossa havaittuja metaboliitteja se ei tuottanut. Pulegonin ja menthofuraanin glutationikonjugaatteja analysoitiin LC/MS/MS tekniikalla ja N- sekä S-/N- sitoutuneiden glutationikonjugaattien pilkkoutumisspektrit tulkittiin tarkasti ensimmäistä kertaa. Tulokset mahdollistavat furaanirenkaan sisältävistä yhdisteistä syntyvien reaktiivisten metaboliittien tehokkaamman seulonnan. Asyyliglukuronit ovat karboksyylihapporyhmän sisältämien yhdisteiden metaboliitteja, jotka voivat olla reaktiivisia. Asyyliglukuronien vaeltamisen puoliintumisajan ja idiosynkraattisten lääkehaittavaikutusten välillä havaittiin selvä korrelaatio. Karboksyylihapporyhmän kanssa voi muodostua myös asyyli koentsyymi A konjugaatteja, jotka voivat olla reaktiivisempia kuin vastaavat asyyliglukuronit. Koentsyymi A ja siitä edelleen syntyviä muita konjugaatteja havaittiin pääasiassa lääkeaineille, joiden todennäköisyys aiheuttaa idiosynkraattisia lääkehaittavaikutuksia oli suurempi.

>Magister Scientiae - MSc / Drought stress is a major contributor to reduced soybean crop yield and quality, this can however be mitigated by the plant’s antioxidant defence mechanisms. One group of antioxidant enzymes that are active in these defence mechanisms are glutathione peroxidases (GPXs). GPXs are antioxidant proteins which are able to reduce H2O2, a toxic reactive oxygen species which accumulates under stress conditions. This study aims at isolating the protein encoded by Glyma01g42840 and determining if it has Phospholipid hydroperoxidase glutathione peroxidase (PHGPX) and/or Thioredoxin dependent peroxidase (TRX-PX) activity as well as assaying the effect of Drought stress on the expression of this putative GPX . This will be accomplished by molecular cloning, sequencing as well as the expression of the isolated protein to assay it enzymatic activity. It was found that the enzyme encoded by Glyma01g42840 is able to use glutathione and thioredoxin as electron donors for the detoxification peroxides, however enzymatic activity is more efficient when using glutathione as an electron donor. In conclusion it was found that glyma01g42840 encodes an enzyme which is able to utilise more than one electron donor and as glutathione produces the greatest amount of enzymatic activity it can be said that glyma01g42840 encodes a GPX.

Le vieillesse conduit à une perte de masse et de fonctionnalité musculaire : la sarcopénie. Ce phénomène physiologique est reconnu comme une cause directe de fragilité et ces deux syndromes gériatriques ont été reliés à de nombreux facteurs dont le statut nutritionnel et la médication. Cependant, les interactions médication-muscle n'ont été que peu étudiées notamment au cours du vieillissement. Le médicament le plus prescrit et consommé est le paracétamol dont la détoxification hépatique nécessite un nutriment : la cystéine (Cys)[...]Sous réserve de la validité de ces résultats chez l'homme, les données obtenues indiquent qu'il serait d'intérêt de mettre en place des recommandations nutritionnelles basées sur une augmentation en Cys au cours des périodes de traitement.Ceci permettrait d'accroître le bénéfice /risque du paracétamol, qui est largement prescrit en première intention pour traiter les douleurs chroniques d'intensité faible à modérée chez les personnes âgées. / The age-related muscle mass and functionality loss, named sarcopenia is a physiological processknown as a direct cause of frailty. Both geriatrics syndromes have been correlated to various factorsas nutritional status and medication. However, the interactions between medication and muscle havenot being deeply investigated, especially in elderly. Using this model, we have highlighted a 12% loss of muscle mass in old rat under repeatedparacetamol cures. This loss was associated with paracetamol-induced sulfur amino acid disturbancesleading to altered glutathione (GSH a Cys containing tripeptide) status and circulating Cys. All together, these results emphasize that muscle mass loss induced by repeated cures withparacetamol in old rats arise from the increased protein and more specifically Cys requirements. Therelevance of these results for older persons needs to be further evaluated before considering whetherit it could be of interest to develop nutritional recommendations aimed to optimized protein intake orCys supplementation during cures with paracetamol. That could further increase the benefit/risk ratioof paracetamol which is widely prescribed for pain relief, especially in older people.

Outre son role majeur dans la resistance cellulaire au stress oxydant, le tripeptide glutathion (L-ƒÁ glutamyl- cysteinyl-glycine) est essentiel a la survie cellulaire. Un deficit en glutathion, associe a un stress oxydant, est un trait commun a plusieurs maladies chroniques inflammatoires et degeneratives. Dans le cadre de ces differentes pathologies, plusieurs etudes ont montre que la prise orale de N-acetylcysteine (NAC), un precurseur de glutathion, ameliorait l'etat des patients. Le stress oxydant et l'inflammation sont deux caracteristiques principales de l'insuffisance cardiaque et des dystrophies musculaires. Nous avons pose l'hypothese d'un deficit en glutathion dans ces maladies et les benefices possibles d'un traitement par le NAC. Dans le modele du rat developpant une insuffisance cardiaque post-infarctus, nous montrons qu'il existe un deficit en glutathion tissulaire. Un mois de traitement oral par le NAC, donne en curatif post-infarctus, restaure le taux de glutathion cardiaque, reduit le stress oxydant, et interrompt le cycle vicieux inflammation/mort cellulaire, TNF/TNF-R1/NSMase/ caspase-3/apoptose. Un deficit en glutathion systemique et tissulaire caracterise aussi les souris LmnaH222P/H222P de 6-7 mois developpant une cardiomyopathie dilatee, modele de la cardiomyopathie associee a la dystrophie musculaire dfEmery Dreifuss. Un mois de traitement oral par le NAC reduit chez les souris de 7 mois la dilatation ventriculaire gauche et la dysfonction contractile, limite la progression de la fibrose cardiaque et lfinflammation. Ceci est associe a une repletion en glutathion et une normalisation de l'expression des enzymes du metabolisme du glutathion, a une diminution du stress oxydant et de l'expression du TNF. Une premiere etude chez les patients de chirurgie cardiaque (n=91) nous a permis de mettre en evidence que : d'une part, il existait un deficit en glutathion auriculaire chez les patients de la classe NYHA IV compares aux patients de la classe NYHA I. D'autre part, les patients asymptomatiques (classe NYHA I) presentaient un deficit en glutathion sanguin, compares aux individus sains, aggrave chez les patients symptomatiques (classes NYHA II a IV). Le deficit en glutathion sanguin chez les patients asymptomatiques precede lfelevation du taux sanguin de TNFR1, un marqueur standard du degre de severite de l'insuffisance cardiaque. Une seconde etude, chez des patients porteurs dfune mutation de la lamine (n=28) et susceptibles de developper une cardiomyopathie dilatee d'Emery Dreifuss, montre que certains de ces patients presentent un deficit en glutathion sanguin, associe chez un seul de ces patients a un taux eleve de TNFR1. L'analyse comparee des donnees biochimiques et cliniques est en cours. Les souris DMDmdx4cv sont un modele experimental de la dystrophie musculaire de Duchenne (DMD), mais ne developpent que tardivement la maladie cardiaque. Nous observons une augmentation du taux du glutathion systemique chez les souris de plus de 10 semaines. Un traitement oral avec un inhibiteur de synthese du glutathion a faible dose, le Lbuthionine sulfoximine (5 mM BSO), ramene le taux de glutathion systemique chez la souris DMDmdx4cv au taux chez la souris sauvage, mais provoque des alterations des cardiomyocytes identifiees par immunohistochimie, des micronecroses, des anomalies de capillaires et des anomalies mitochondriales observees en microscopie electronique. En conclusion, le deficit en glutathion est un evenement precoce et durable au cours de l'insuffisance cardiaque, d'origine ischemique ou genetique. Les perspectives offertes par ces resultats sont: 1) le test du glutathion sanguin pour le depistage de sujets asymptomatiques a risque; 2) l'indication de NAC aux patients cardiaques, en complement du traitement courant / The tripeptide glutathione (L-? -glutamyl-cysteinyl-glycine) does not only play a major in cellular resistance to oxidative stress, but is also essential to cell survival. A deficit in glutathione, associated with oxidative stress, is a common hallmark of several chronic inflammatory and neurodegenerative diseases. In different examples, several studies reported that oral treatment with N-acetylcysteine (NAC), a glutathione precursor, improved patient status. Oxidative stress and inflammation are two main characteristics of heart failure (HF) and muscular dystrophy. We hypothesized that glutathione deficiency occurred in these diseases and that treatment with NAC might be beneficial. In post-myocardial infarction (MI) rats, with established chronic HF, we show that cardiac tissue is depleted in glutathione. Curative 1-month oral NAC treatment replenishes cardiac tissue glutathione, reduces oxidative stress and disrupts the vicious inflammation/cell death, TNF/TNF-R1/N-SMase/ caspase-3/ apoptosis cycle. Deficit in serum and cardiac glutathione also characterize 6- to 7-month old LmnaH222P/H222P mice with dilated cardiomyopathy (DCM), a model of the cardiomyopathy associated with Emery Dreifuss muscular dystrophy (EDMD), One-month oral NAC treatment reduces left ventricular dilation and contractile dysfunction, limits the progression of cardiac fibrosis and inflammation in 7-month old LmnaH222P/H222P mice. This is associated with glutathione repletion and normalization of glutathione metabolism enzymes, and reduction of oxidative stress and TNF expression. In a first study in cardiac surgery patients (n=91) we show that: on the one hand, atrial glutathione is depleted in patients of NYHA class IV compared with asymptomatic patients of NYHA class I. On the other hand, asymptomatic patients of NYHA class display a deficiency in blood glutathione compared with healthy controls that worsens in asymptomatic patients of NYHA class II-IV. Blood glutathione deficiency in asymptomatic patients precedes elevation of blood TNFR1, a standard marker of HF severity. A second study, in patients with lamin mutation (n=28), prone to develop an Emery Dreifuss DCM, shows that a number of patients display blood glutathione deficiency, with one patient having a high blood TNFR1 level. Analysis of clinical data is underway. DMDmdx4cv mice are an experimental model of Duchenne muscular dystrophy. We observe an increase in blood glutathione in 10-week-old mice and older. Oral treatment with a low dose of L-buthionine sulfoximine (5 mM BSO), an inhibitor of glutathione synthesis, resumes blood glutathione in DMDmdx4cv mice to the control value in WT mice, but produces alterations in cardiomyocytes identified by immunohistochemistry and micronecrosis, capillary and mitochondrial abnormalities observed by electronic microscopy. In conclusion, glutathione deficiency is an early and lasting event in ischemic or genetically-linked HF. These results pave the way for two possible applications: 1) blood glutathione test for the screening of asymptomatic individuals at risk for HF; 2) NAC indication to cardiac patients in addition to current treatment

Glutathione syntethase (GS) is an enzyme that belongs to the ATP-grasp superfamily and catalyzes the second step in the biosynthesis of glutathione. GS has been purified and sequenced from a variety of biological sources; still, its exact mechanism is not fully understood. Four highly conserved residues were identified in the binding site of human GS. Additionally, the G-loop residues that close the active site during catalysis were found to be conserved. Since these residues are important for catalysis, their function was studied computationally by site-directed mutagenesis. Starting from the reported crystal structure of human GS, different conformations for the wild type and mutants were obtained using molecular dynamics technique. The key interactions between residues and ligands were detected and found to be essential for enzyme activity.