NAD(P)H:Quinone oxidoreductase-1 (NQO1) has been implicated in the bioreductive activation of the clinically active anticancer drug Mitomycin C (MMC) and a polymorphic variant of NQO1 which lacks functional enzyme activity (NQO1*2) has been linked with poor survival in patients treated with MMC. The relationship between NQO1 activity and cellular response to MMC is however controversial and the aim of this study was to determine whether the response of bladder cancer patients to MMC can be forecast on the basis of NQO1*2 genotype status. Genomic DNA was extracted from formalin-fixed, paraffin-embedded tissue from 148 patients with low to intermediate grade (G1/G2) superficial (Ta/T1) bladder cancers and NQO1*2 genotype status determined by PCR-RFLP. NQO1*2 genotype status was retrospectively compared with clinical response to intravesical administered MMC with the primary end-point being time to first recurrence. NQO1 phenotype was determined by immunohistochemistry. Of the 148 patients genotyped, 85 (57.4%) were NQO1*1 (wild-type), 59 (39.8%) were NQO1*1/*2 (heterozygotes) and 4 (2.7%) were NQO1*2/*2. No NQO1 protein expression was detected in NQO1*2/*2 tumours. A broad spectrum of NQO1 protein expression existed in tumours genotyped as NQO1*1 and NQO1*1/*2 although tumours with NQO1*1 typically expressed higher NQO1 protein. A poor correlation existed between NQO1*2 genotype status and clinical response to MMC. The results of this retrospective study suggest that tailoring MMC therapy to individual patients with superficial bladder cancer on the basis of NQO1 genotype status is unlikely to be of clinical benefit.

Aristolochic acids (AA) are carcinogenic and nephrotoxic alkaloids from Aristolochia species. Aristolochic acid I (AAI), the major component of AA, causes the development of Aristolochic acid nephropathy (AAN) and Balkan endemic nephropathy (BEN). These two diseases cause total renal failure and urothelial malignancies. The fact that these diseases have not been developed in all persons, who have been exposed to their action, might be causd by different activities and protein levels of the enzymes metabolizing AAI. Thus, the identification of enzymes involved in the metabolism, and detailed knowledge of their expression and catalytic specifities is a major importance. Aristolochic acid I (AAI) can be metabolized by several types of reactions. Like most nitroaromatics, the main activation pathway of AAI is reduction of its nitro group to form a cyclic acylnitrenium ion, which can bind to the purine bases, thereby forming AAI-DNA adducts. The detoxication pathway of AAI is its oxidative demethylation by cytochromes P450 forming detoxication metabolite 8-hydroxyaristolochic acid Ia (AAIa). In the present thesis, using rat and human enzymes and as well as several mice models, the metabolism of AAI in vitro and in vivo was investigated. The first model has deleted gene for NADPH:cytochrome P450...

No / NAD(P)H:quinone oxidoreductase-1 (NQO1) is a potential target for therapeutic intervention but attempts to exploit NQO1 using quinone-based bioreductive prodrugs have been largely compromised by toxicity to organs that inherently express high levels of NQO1. In an attempt to circumvent this problem, this study describes the development of a tripartite quinone-based drug delivery system, the ultimate objective of which is to release a targeted therapeutic agent following the reduction of a quinone "trigger" by NQO1. Molecular modeling of drug/NQO1 interactions were conducted prior to the synthesis of N-{4-[bis-(2-chloroethyl)-amino]-phenyl}-beta,beta,2,4,5-pentamethyl-3,6-dioxo-1,4-cyclohexadiene-1-propanamide (prodrug 1). Prodrug 1 is a good substrate for purified NQO1 (V(max) and K(m) values of 11.86 +/- 3.09 micromol/min/mg and 2.70 +/- 1.14 micromol/L, respectively) and liquid chromatography-mass spectrometry analysis of the metabolites generated showed that lactone 3 and aniline mustard 4 were generated in a time- and NQO1-dependent manner. Chemosensitivity studies showed that prodrug 1 is selectively toxic to cells that overexpress NQO1 under aerobic conditions, and comet assay analysis confirmed the presence of elevated interstrand cross-links in NQO1-rich compared with NQO1-deficient cells. Hypoxic sensitization (hypoxic cytotoxicity ratio = 15.8) was observed in T47D cells that overexpress cytochrome P450 reductase. In conclusion, the results of this study provide mechanistic proof of principle that a tripartite benzoquinone drug delivery system is enzymatically reduced to release an active therapeutic agent. Further development of this concept to fine-tune substrate specificity for specific reductases and/or the inclusion of alternative therapeutic agents is warranted.

In unseren Studien haben wir uns unter Verwendung von in vitro und in vivo Modellen des neuronalen Zelltodes mit verschiedenen neuroprotektiven Mechanismen beschäftigt. Uns interessierten Faktoren aus drei verschiedenen Komplexen, die an zellulärer Vitalität teilhaben. Im Mittelpunkt unserer Untersuchung stand ein antioxidatives Enzym und dessen Rolle bezüglich des neuronalen Zelltodes - die NAD(P)H-Quinonoxidoreduktase1. In den Sudien haben wir die Aktivität, Expression und die Lokalisation dieses Enzyms nach neuronalem Schaden untersucht. Wir konnten zeigen, dass die Quinonoxidoreduktase den neuronalen Schaden exazerbieren kann. Ein anderer Schwerpunkt unserer Arbeit bildete das Steroidhormon 17-beta-Estradiol und sein neuroprotektiver Einfluss auf neurodegenerative Prozesse. Wir deckten verschiedene Expressionen der beiden Estradiolrezeptoren alpha und beta in kortikalen, septalen und hippokampalen Nervenzellen auf und das daraus folgende unterschiedliche neuroprotektive Potenzial des Hormons. Im dritten Teil der Studien konnten wir den Einfluss des zytoskelettmodulierenden Proteins Gelsolin auf die Progression des neuronalen Zelltodes nach zellulärem Schaden charakterisieren. / In our studies we characterized different neuroprotective mechanism by in vitro and in vivo models of neuronal cell death. We were interested in factors of three different complexes wich play a role in cellulare vitality. In the centre of our studies there was an antioxidative enzyme and his role in the neuronal cell death-the NAD(P)H:quinone oxidoreductase1. We have investigated in the activity, expression and localisation of this enzyme after neuronal damage. We could show that this enzyme can exacerbate neuronal cell death. Another point of our work were the steroid hormon 17-beta-Estradiol and the neuroprotective character in neurodegeneration. We discovered different expression of the two estradiol- receptors alpha and beta in brain caused in different protective potential to cortical, septal and hippocampal structures by the hormon. The cytoskeletal structures modulate by the protein gelsolin was the third part of the studies. We could show that the modulation of the cytoskeleton were involved in the progression of neuronal cell death after cellulare damage.

This Ph.D. thesis addresses the structure-function relationship of the multimeric oxidoreductase WrbA with the principal aim being the explanation of the unusual kinetics of this enzyme in molecular terms, and thus getting an insight about its physiological role in bacteria. WrbA is a multimeric enzyme with FMN as a co-factor, catalyzing the oxidation of NADH by a two electrons transfer. Structure and function analysis of WrbA places this enzyme between bacterial flavodoxins and eukaryotic oxidoreductases in terms of its evolutionary relationship. The kinetic activity of WrbA was studied under varying conditions such as temperature, pH etc, and its kinetic mechanism was evaluated from parameters KM and Vmax and confirmed by product inhibition pattern experiments. Crystallization and proteolytic experiments also underpin the functional importance of the multimeric nature of WrbA and aid the understanding of the physiological role of this enzyme in molecular terms.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2012-08-29T21:11:40Z No. of bitstreams: 1 Silvana Sousa Paz Avaliação de marcadores....pdf: 756990 bytes, checksum: 5aac886be232eac44d86b25a30837ac4 (MD5) / Made available in DSpace on 2012-08-29T21:11:40Z (GMT). No. of bitstreams: 1 Silvana Sousa Paz Avaliação de marcadores....pdf: 756990 bytes, checksum: 5aac886be232eac44d86b25a30837ac4 (MD5) Previous issue date: 2012 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, Bahia, Brasil / As leucemias são malignidades hematopoiéticas, caracterizadas por subgrupos biologicamente distintos, sendo os tipos mais frequentes de cânceres em crianças e adolescentes. Polimorfismos em genes de enzimas que metabolizam xenobióticos podem estar relacionados com a inserções/deleções, polimorfismos de nucleotídeo simples (SNP’s) e variações no número de cópias e têm sido relacionados com a patogênese de algumas neoplasias hematológicas, como a leucemia linfóide aguda (LLA). O objetivo deste estudo foi o de determinar as frequências de polimorfismos em genes associados ao estresse oxidativo e metabolismo de xenobióticos (GSTT1, GSTM1, CYP2E1, NQO1 e MPO), em pacientes pediátricos com LLA, associando-as a aspectos clínicos e marcadores de evolução da doença. A casuística foi composta por 37 pacientes pediátricos seguidos na clínica ONCO e tratados pelo protocolo GBTLI-LLA 93. O perfil hematológico dos pacientes foi realizado ao diagnóstico e durante o tratamento e os polimorfismos gênicos foram investigados por reação da polimerase em cadeia - polimorfismo de tamanho de fragmento de restrição (PCR-RFLP) e por reação da polimerase em cadeia multiplex (PCR Multiplex). As análises estatísticas apresentaram significância para os valores de leucócitos totais nos D1 e D7 (p= 0,0016) e nos D1 e D14 (p= 0,0059); linfócitos nos D1 e D7 (p= 0,0088) e D1 e D14 (p= 0,0101); segmentados neutrófilos nos D1 e D7 (p= 0,0033) e D1 e D14 (p= 0,0252); blastos periféricos D1 e D7 (p< 0,0001) e D1 e D14 (p< 0,0001) e; para a contagem de blastos na medula óssea (MO) nos D1 e D15 (p<0,0001), D1 e D28 (p< 0,0001) e D15 e D28 (p= 0,0005). As frequências alélicas e genotípicas para os genes estudados estavam em equilíbrio de Hardy-Weinberg. A mutação do gene MPO foi associada a infiltração da MO (p= 0,0473) e presença de blastos no líquor (p= 0,0473). O polimorfismo do gene GSTT1 foi associado à contagem de leucócitos (p= 0,014) e plaquetas (p= 0,0034) no D1 e a contagem de leucócitos (p=0,037) e segmentados neutrófilos (p= 0,0008) no D7. A presença do polimorfismo no gene NQO1 foi associado à infiltração da MO (p= 0,0410) e a presença de blastos no líquor (p= 0,0410). Entretanto, o polimorfismo NQO1 apresentou associação com a presença de palidez (p=0,0096). Os dados encontrados corroboram em parte com dados encontrados na literatura, sendo necessária a realização de um estudo com numero maior de pacientes para confirmação dos achados relacionados aos genes investigados e a LLA. / Leukemia is characterized by biologically distinct subgroups and is the most frequent hematological malignity in childhood. Polymorphisms in genes of enzymes that metabolize xenobiotics may be related to insertions/ deletions, single nucleotide polymorphisms (SNP's) and gene copies variation and have been related to the pathogenesis of some hematologic malignancies, including acute lymphoblastic leukemia (ALL). The aim of this study was to investigate genes polymorphisms associated with the oxidative stress and xenobiotic metabolism (GSTT1, GSTM1, CYP2E1, NQO1 and MPO) in a group of childhood ALL patients, associating them with clinical evolution and prognostic markers. The casuistic was compound by 37 pediatric patients followed and treated at the clinic ONCO with the protocol GBTLI-LLA 93. The hematological profile of patients was performed at diagnosis and during treatment and gene polymorphisms were investigated by Polimerase Chain Reaction - Restriction Fragment Length Polymorfism (PCR-RFLP) and Polimerase Chain Reaction Multiplex (Multiplex PCR). Statistical analyses were significant for values of total leukocytes in D1 and D7 (p= 0.0016) and in D1 e D14 (p= 0.0059); lymphocytes in D1 and D7 (p= 0.0088), D1 and D14 (p= 0.0101); neutrophils in D1 and D7 (p= 0.0033), D1 and D14 (p= 0.0252). It was also find statistical significance at the number of peripheral blasts in D1 and D7 (p< 0.0001), D1 and D14 (p< 0.0001); the blast count in bone marrow (BM) in D1 and D15 (p<0.0001), D1 and D28 (p< 0.0001) and D15 and D28 (p= 0.0005). The allelic and genotypic frequencies of all gene polymorphism investigated were in Hardy-Weinberg equilibrium. The MPO gene mutation was associated with infiltration of the BM in D28 (p= 0.0473) and the presence of blasts in the CSF (p= 0.0473). The GSTT1 gene polymorphism was associated with leukocyte (p= 0.014) and platelet counts (p= 0.0034) in D1 and with leukocytes (p=0,037) and neutrophils counts (p= 0.0008) in D7. The NQO1 gene polymorphism presence was associated with BM infiltration at D28 (p= 0.0410) and the presence of blasts in the CSF (p= 0.0410). However, the NQO1 polymorphism was associated with the presence of pallor (p=0.0096). Result described here corroborated in part with previous described data, being necessary to carry out additional study with a larger number of patients to confirm the finding related to genes polymorphism investigated and the clinical evolution of ALL patients.

Les peroxyrédoxines sont des enzymes essentielles de la cellule. Outre leur rôle d’antioxydant, elles sont aussi des régulateurs de la signalisation cellulaire et des suppresseurs de tumeurs. La péroxiredoxine 1 (Prx1) est la plus abondante parmi les six isoformes de peroxyrédoxines humaines. Elle est fréquemment surexprimée dans plusieurs types de cellules cancéreuses, et on a pu associer Prx1 aux processus de carcinogenèse et de métastase, ainsi qu’à la résistance à la radiothérapie ou la chimiothérapie. Ainsi, Prx1 pourrait donc être une cible anticancéreuse intéressante. Au cours de ce travail de thèse, nous avons d’abord évalué l'impact d’une diminution de Prx1 (Prx1 knockdown (Prx1–)) sur la sensibilité cellulaire à des dizaines de médicaments anticancéreux dont la vinblastine, le taxol, la doxorubicine, la daunorubicine, l’actinomycine D, et le 5-fluorouracile, et d’agents connus pour provoquer la production d’espèces réactives de l’oxygène (ROS), dont le peroxyde d'hydrogène, le 2-phényléthyle isothiocyanate, le β-lapachone (β-lap) et la ménadione. Nous avons mis en évidence qu’une diminution de Prx1 augmente significativement la sensibilité des cellules à l'effet cytotoxique de la β-lap et de la ménadione, deux naphtoquinones possédant une activité anti-tumorale.Nous avons étudié les mécanismes responsables de l'augmentation de la cytotoxicité de la β-lap dans un contexte Prx1–. Nous montrons que la toxicité accrue de la β-lap dans des cellules Prx1– est due à une accumulation intracellulaire de ROS. Cet effet est dépendant de l’activité NADPH quinone oxydoréductase (NQO1) et s’accompagne d’une phosphorylation de c-Jun N-terminal kinases (JNK), protein 38 (p38), extracellular signal-regulated kinases (Erk) et des mitogen-activated protein kinases (MAPK), mais aussi d’une diminution des niveaux protéiques de la thiorédoxine 1. En se basant sur le fait que Prx1 est une enzyme antioxydante et un partenaire d'au moins ASK1 et JNK, deux éléments clés de la voie MAPK, nous proposons que la sensibilisation à la β-lap, observée après diminution de Prx1, est provoquée par une action synergique entre l'accumulation de ROS et l'induction de la voie MAPK, conduisant ainsi à l'apoptose.Nous avons ensuite étudié les mécanismes responsables de l'augmentation de la cytotoxicité de la ménadione dans le contexte Prx1–. La sensibilité accrue des cellules à l'effet cytotoxique de la ménadione et également associée à l'accumulation rapide et massive des ROS intracellulaire et à une mort cellulaire ressemblant à la nécrose programmée (necroptosis). L’accumulation de ROS induite par la ménadione et très rapidement détectée dans le cytosol, le noyau, et de façon encore plus importante, dans la matrice mitochondriale. Ce phénomène est en corrélation avec l'oxydation importante des thiorédoxine 2 et peroxiredoxine 3, deux protéines antioxydantes localisées dans la mitochondrie. La diminution de l’expression de Prx1 s’accompagne d’une augmentation des quantités tant de l’ARNm que de la protéine NRH: quinone oxydoréductase 2 (NQO2). Cette augmentation de l'activité de NQO2 est en grande partie responsable de l'accumulation intracellulaire de ROS et de la mort cellulaire après le traitement à la ménadione. Nos données révèlent que l’accumulation de ROS dans les cellules Prx1– provient de la résultante entre l’augmentation de leur production par NQO2 au cours du métabolisme de la ménadione et la diminution de leur élimination par Prx1. Enfin et de façon surprenante, selon la nature des naptoquinones (β-lap ou ménadione), les voies métaboliques qui conduisent à l'accumulation des ROS, ou les voies de signalisation et les mécanismes de mort cellulaire impliqués semblent être distincts. / Peroxiredoxins have multiple cellular functions as major antioxidants, signaling regulators, molecular chaperones and tumor suppressors. Peroxiredoxin 1 (Prx1) is the most abundant among the six isoforms of human peroxiredoxins. It is frequently over-expressed in various cancer cells, which is known associated with carcinogenesis, metastasis and resistance to radiotherapy or chemotherapy. Prx1 could thus be an interesting anticancer target. In this study, we first evaluated the impact of Prx1 knockdown (Prx1–) on cellular sensitivity to dozens of anticancer drugs including vinblastine, taxol, doxorubicin, daunorubicin, actinomycin D, and 5-fluorouracil, and of reactive oxygen species (ROS)-generating agents, including hydrogen peroxide, 2-phenylethyl isothiocyanate, β-lapachone (β-lap) and menadione. We observed that Prx1 knockdown significantly enhanced cancer cell sensitivity to β-lap and menadione, two naphthoquinones with anti-cancer activity.We first investigated the underlying mechanisms responsible for the specifically enhanced cytotoxicity to β-lap in a Prx1 knockdown context. Prx1 knockdown markedly potentiated β-lap-induced cytotoxicity through ROS accumulation. This effect was largely NAD(P)H:quinone oxidoreductase 1 (NQO1)-dependent and associated with the phosphorylation of c-Jun N-terminal kinases (JNK), protein 38 (p38) and extracellular signal-regulated kinases (Erk) proteins in mitogen-activated protein kinase (MAPK) pathways, and a decrease in thioredoxin 1 protein levels. Based on the fact that Prx1 is a major ROS scavenger and a partner of apoptosis signaling kinase 1 (ASK1) and JNK, two key components of MAPK pathways, we propose that Prx1 knockdown-induced sensitization to β-lap is achieved through the combined action of ROS accumulation and MAPK pathway activation, leading to cell apoptosis.We then investigated the underlying mechanisms responsible for the specifically enhanced cytotoxicity to menadione in Prx1– cells. Enhanced sensitivity to menadione was associated with a rapid and significant intracellular ROS accumulation and necroptotic-like cell death. Menadione-induced ROS accumulation occurred immediately in the cytosol, the nucleus, and even more noticeably in the mitochondrial matrix, correlated with significant oxidation of both mitochondria-localized thioredoxin 2 and peroxiredoxin 3. Prx1 knockdown significantly up-regulated mRNA and protein levels of NRH: quinone oxidoreductase 2 (NQO2). Increased activity of NQO2 was largely responsible for menadione-induced ROS accumulation and consequent cell death. Our data indicate that massive ROS accumulation results from the combined effect of increased ROS generation by higher NQO2 activity during menadione metabolism, and diminished Prx1 scavenging activity. Finally and noteworthy, the metabolic pathways that lead to ROS accumulation, downstream signaling pathways and cell death mechanisms appear to be distinct for β-lap and menadione.