Les Formes Réactives de l'Oxygène (FRO) regroupent des molécules comme les radicaux superoxide ($O_2^{\bullet -}$)et hydroxyle ($HO^\bullet$) ou le peroxyde d'hydrogène ($H_2O_2$) produites au sein des cellules en aérobiose. Malgré des systèmes de défense, des FRO peuvent réagir fortuitement avec des protéines, des lipides ou l'ADN provoquant des dommages cellulaires dont les mécanismes ne sont pas encore entièrement élucidés. Afin d'appréhender ce``stress oxydant'', cette thèse présente des simulations numériques de la dynamique de FRO en utilisant la bactérie E. coli comme organisme modèle. Dans un premier temps, les simulations numériques sont réalisées de façon déterministe sur un ensemble de cellules. L'étude de la mortalité de E. coli exposé à $H_2O_2$ montre que le fer intracellulaire libre et la densité cellulaire, deux facteurs potentiellement impliqués dans la dynamique des FRO, jouent un rôle primordial dans l'interprétation expérimentale comme par exemple le comportement bi-modal de E. coli opposé à $H_2O_2$. Nous avons également évalué les rôles relatifs des principales défenses mises en place contre $H_2O_2$ à savoir la membrane cellulaire et les enzymes. Une étude détaillée indique que leur implication dépend non linéairement de la concentration en $H_2O_2$.Dans une seconde approche nous réduisons l'échelle d'étude pour nous ramener à la cellule unique dans les conditions physiologiques. Il apparaît ainsi que la stochasticité intrinsèque des réactions chimiques associées aux FRO permet à certaines bactéries de se différencier en vue d'un futur stress. / The Reactive Oxygen Species (ROS) are molecules (superoxide $O_2^{\bullet -}$, hydrogen peroxide $H_2O_2$ and hydroxyl radical $HO^\bullet$) generated in living cells as a consequence of aerobic life. They are partially eliminated by scavenging systems. Nevertheless, ROS can unfortunately react with cellular proteins, lipids or DNA leading to cell damage. The mechanisms of such lesions is still being studied: we are talking about``oxidative stress''. Using Escherichia coli as a model organism this thesis is concerned with the numerical simulation of ROS dynamics.In the first part of this work, simulations were performed in a deterministic way to predict the behaviour of a set of cells. By studying killing of E. coli by exposure to $H_2O_2$, we show that intracellularavailable iron and cell density, two factors potentially involved in ROSdynamics, play a major role in the prediction of experimental results in particular in bimodal cell killing.We then evaluate the relative roles of major defences against $H_2O_2$. Although the key actors in celldefence are enzymes and membrane, a detailed analysisshows that their involvement depends on the $H_2O_2$ concentration level. In the second part, we study more closely the fate of the single cell with a stochastic point of view in physiological conditions.We show that elementary chemical stochasticity allows bacteria to segregate specialized cells in prevision of possible stress challenge. Actually, whereas ROS distribution does not activate defence regulation without exogenous stress, we demonstrate that this distribution may activate DNA repair mechanisms.

Este trabalho apresenta dados relativos a oxidação de melatonina catalisada por peroxidase de rábano (HRP, horseradish peroxidase) e mieloperoxidase (MPO). Em presença de peróxido de hidrogênio (H2O2), HRP catalisa a oxidação de melatonina com formação de um produto de clivagem do anel indólico, a N1-acetil-N2-formil-5-metoxiquinuramina. Esta reação consome oxigênio e apresenta quimiluminescência na região de 440-540 nm. Quimiluminescência e a formação da quinuramina também foram observados quando HRP/H2O2 foram substituídos por neutrófilos ativados por acetato de forbol miristato ou zimosan opsonizado. Em neutrófilos, tanto a emissão de luz quanto a formação do produto foram inibidos pela adição de azida, um inibidor de MPO. Superóxido dismutase tem um forte efeito inibidor sobre a emissão de luz enquanto que catalase e ácido úrico não apresentam qualquer efeito. A oxidação de melatonina por neutrófilos ativados pode ser relevante in vivo e estar associada com algumas das funções descritas para mieloperoxidase e melatonina. A possível implicação biológica da oxidação de melatonina por neutrófilos, especialmente em condições inflamatórias, é discutida. / In the presence of hydrogen peroxide, horseradish peroxidase (HRP) catalyzes the production of N1-acetyl-N2-formyl-5-methoxykynuramine from melatonin. This reaction consumes oxygen and exhibits chemiluminescence in the 440-540 nm region. The excited cleavage product derived from the thermolysis of an intermediate dioxetane is suggested to be the emitting species. Chemiluminescence and the indole ring cleavage product were also observed when HRP/H2O2 was replaced by phorbol myristate acetate or opsonized zymosan-activated neutrophils. Azide, a myeloperoxidase inhibitor, strongly suppressed melatonin oxidation. Superoxide dismutase has a strong inhibitory effect on light emission but catalase and uric acid are without effect on the emission. The oxidation of melatonin by activated neutrophils may be relevant to the in vivo functions of myeloperoxidase and melatonin. The possible biological implication of melatonin oxidation by neutrophils, especially in inflammatory conditions, is discussed.

A caprinocultura no Brasil vem crescendo consideravelmente nos últimos anos. No entanto, este crescimento é limitado pela qualidade espermática pós-descongelamento, o que inviabilizaria a utilização de biotecnologias aplicadas à reprodução (e.g., inseminação artificial e transferência de embriões). Um dos motivos para a queda na qualidade espermática pós-descongelação é o ataque das espécies reativas de oxigênio (ROS), que podem levar a danos em membrana, acrossomo, mitocôndria e DNA. Uma alternativa para a melhora na qualidade espermática de amostras criopreservadas de caprinos seria o tratamento do meio diluidor com a antioxidantes. No entanto, é fundamental identificar a espécie reativa de oxigênio mais deletéria ao sêmen de caprinos para determinar o antioxidante ideal. O objetivo do presente estudo foi identificar a ROS mais deletéria ao sêmen de caprinos e, com isso, tratar o diluidor com antioxidantes específicos para a destruição da mesma. No experimento 1, amostras espermáticas de 12 bodes foram submetidas á incubação com 3 sistemas de produção de ROS e um subproduto da peroxidação de lipídeos, conhecida por também levar a danos oxidativos. As amostras espermáticas foram submetidas a incubação com xantina (20mM) e xantina oxidase (ânion superóxido), sulfato de ferro (4mM) e ascorbato (20mM- Radical hidroxila), peróxido de hidrogênio (H2O2; 20 mM) e malondialdeído (20 mM). Após a incubação as amostras espermáticas foram avaliadas quanto a morfologia, motilidade, membrana (eosina/nigrosina), acrossomo (Rosa Bengala/ Fast Green), mitocôndria (3,3 diaminobenzidina) e substâncias reativas ao ácido tiobarbitúrico (TBARS, avalia a susceptibilidade do espermatozóide ao estresse oxidativo). Os resultados do experimento 1 indicaram que a ROS mais deletéria ao espermatozóide caprino é o H2O2, que levou a danos de membrana plasmática e mitocôndria. Assim, no Experimento 2, o diluidor para a criopreservação do sêmen caprino foi suplementado com catalase e Glutationa Peroxidase (GPx), antioxidantes enzimáticos responsáveis pela destruição do H2O2. A análise estatística dos dois experimentos foi realizada através do SAS system for Windows. Os resultados do experimento 2 não revelaram efeitos benéficos para nenhuma das variáveis e com a utilização de ambos os antioxidantes. De fato, a catalase apreesntou efeitos deletérios à membrana plasmática das amostras criopreservadas com 240 UI/mL de catalase quando comparadas ao controle (13,42±2,96% e 25,08±4,80% de espermatozóides com membrana íntegra, respectivamente). De fato, estudos anteriores indicam que as concentrações de catalase em sêmen de caprinos são extremamente baixas, o que indicaria que o tratamento com as dosagens utilizadas de catalase pode ter sido excessivo. Além disto, diferentes antioxidantes aparentemente atuam em diferentes regiões do espermatozóide, sendo que, apenas a combinação de diferentes antioxidante poderia ser eficiente. Por outro lado, os resultados do Experimento 1 foram obtidos em amostras frescas, sendo que provavelmente, em amostras criopreservadas, a espécie reativa mais deletéria poderia ser outra. / The goat production in Brazil has grown considerably in recent years. However, this growth is limited by the post-thaw sperm quality, which would impais the use of biotechnologies applied to reproduction (e.g., artificial insemination and embryo transfer). One reason for the decline in sperm quality after thawing is the attack of reactive oxygen species (ROS), which can lead to damage of membrane, acrosome, mitochondria and DNA. An alternative to improve the quality of cryoprerved goat sperm samples would be the treatment of the extender with antioxidants. However, it is crucial to identify the reactive oxygen species more deleterious to semen of goats to determine the ideal antioxidant. The aim of this study was to identify the ROS more deleterious to semen of goats and thereby to treat the extender with specific antioxidants for the destruction of this ROS. In experiment 1, sperm samples from 12 goats were incubated with 3 systems of production of ROS and a by-product of lipid peroxidation, known to also lead to oxidative damage. The sperm samples were subjected to incubation with xanthine (20 mM) and xanthine oxidase (superoxide anion), iron sulfate (4 mM) and ascorbate (20mM-hydroxyl radical), hydrogen peroxide (H2O2, 20 mM) and malondialdehyde (20 mM). After incubation the samples were evaluated for sperm morphology, motility, membrane (eosin / nigrosin), acrosome (Rose Bengal / Fast Green), mitochondria (3.3 diaminobenzidine) and thiobarbituric acid reactive substances (TBARS, assesses the susceptibility of sperm to oxidative stress). The results of experiment 1 indicated that ROS most deleterious to the goat sperm is the H2O2, especially due to damages to the plasma membrane and mitochondria. Thus, in Experiment 2, the extender for cryopreservation of goat semen was supplemented with catalase and glutathione peroxidase (GPx), enzymatic antioxidants responsible for the destruction of H2O2. Statistical analyses were performed using the SAS system for Windows. The results of experiment 2 revealed no beneficial effects of the use of neither catalase oor GPx. In fact, catalase showed deleterious effects on the plasma membrane of cryopreserved samples; Samples treated with 240 IU / mL of catalase showed a lower percentage of sperm with intact membrane when compared with controls (13.42 ± 2.96 ± 4.80% and 25.08%, respectively ). In fact, previous studies indicate that concentrations of catalase in semen of goats are very low, which indicates that catalase treatment with the dosages used in the present study may have been excessive. Apparently different antioxidants work in different regions of the sperm, and only the combination of different antioxidant could be effective. Moreover, the results of Experiment 1 were obtained in fresh samples, and probably, in cryopreserved samples, other ROS could be the most deleterious.

Microcistinas (MCs) são heptapeptídeos cíclicos produzidos por cianobactérias e possuem potente hepatotoxicidade e atividade promotora de tumor. Em intoxicações agudas induzidas por MCs ocorre infiltração leucocitária no foco inflamatório. Embora os mecanismos de hepatotoxicidade não são claros, o recrutamento de neutrófilos no fígado pode contribuir ao dano tecidual e desenvolvimento tumoral causados por xenobióticos. O objetivo dessa tese foi investigar os efeitos de três estruturalmente distintas MCs (MC-LA, MC-YR e MC-LR) nas seguintes funções de neutrófilos: síntese e expressão de moléculas de adesão, rolamento, adesão, migração e liberação de citocinas e de ROS. Nos ensaios de migração em bolsa de ar, as três MCs induziram similarmente a migração de leucócitos in vivo em tecido subcutâneo de ratos e diferencialmente a secreção de citocinas pró-inflamatórias (CINC, IL-1β, TNF-α, VEGF-α e MIP) no exsudato. Concentrações elevadas de CINC-2αβ foram encontradas nos exsudatos inflamatórios de animais após injeção de MC-LA, MC-LR ou MC-YR. MIP-2 elevou-se apenas em exsudatos de animais expostos a MC-LR. Não foram observadas alterações em secreção de IL-1β, TNF-α e VEGF-α. Estudos de microscopia intravital mostraram que apenas a aplicação tópica de MC-LR reforçou o rolamento e a adesão de leucócitos no endotélio de vênulas mesentéricas pós-capilares. Esses últimos resultados podem ser dependentes de aumento e expressão da síntese da L-selectina e β2-integrina nos neutrófilos, tal como avaliado por FACS e PCR-RT, respectivamente. Adicionalmente, em ensaios em câmara de Boyden in vitro, as três MCs promoveram locomoção direta de neutrófilos e aumentaram a migração dessas células em resposta ao fMLP, além do aumento de cálcio intracelular observado por microscopia confocal. Os efeitos das MC-LA, MC-YR e MC-LR em neutrófilos humanos e de ratos tiveram o mesmo padrão de resposta. A análise de viabilidade celular, fragmentação de DNA, despolarização de membrana mitocondrial e liberação de ROS intracelulares foram avaliadas pela técnica de FACS. A concentração de ROS extracelular foi medida por quimiluminescência amplificada por lucigenina. A produção de citocinas em células tratadas foram determinadas por ELISA. Observamos aumento da liberação de IL-8, CINC-2α β e da concentração de ROS extracelular por neutrófilos humanos e de ratos pela exposição dessas células com as MCs. Os resultados aqui obtidos mostram o efeito pró-inflamatório direto das MC-LA, MC-YR e MC-LR. A possibilidade de os neutrófilos migrarem e responderem localmente a MCs contribui para a toxicidade destas toxinas. / Microcystins (MCs) are a family of heptapeptide toxins produced by some genera of Cyanobacteria. MCs have potent hepatotoxicity and tumor-promoting activity. Leukocyte infiltration in the liver was observed in MC-induced acute intoxication. Although the mechanisms of hepatotoxicity induced by MCs are still unclear, neutrophil infiltration in the liver may play an important role in triggering toxic injury and tumor development. The purpose of this thesis was to investigate the effects of three structurally distinct MCs (MC-LA, MC-YR and MC-LR) in the neutrophil functions: synthesis and expression of adhesion molecules, rolling, adhesion, migration and release of cytokines and ROS. In migration assays of the air pouch, the three MCs similarly induced the migration of leukocytes in vivo in subcutaneous tissue of rats and differentially the secretion of pro-inflammatory cytokines (CINC-2αβ, IL-1-β, TNF--α, VEGF- α and MIP-2) in exudates. Elevated concentrations of CINC-2αβ were found in the inflamed exudates from animals injected with MC-LA, MC-LR or MC-YR, although MIP-2 was only detected in the exudates from animals injected with MC-LR. There were no changes in the secretion of IL-1-β, TNF-α and VEGF--α. Intravital microscopic studies showed that topical application of MC-LR enhanced the numbers of rolling and adhered leukocytes in the endothelium of postcapillary mesenteric venules. The latter effects may be dependent upon induction of the synthesis and expression of L-selectin and -α2-integrin in neutrophils, as assessed by flow cytometry and RT-PCR, respectively. Conversely, the three toxins promoted direct locomotion of neutrophils and enhanced their migration in response to fMLP, as measured by Boyden chamber assays, and increased intracellular calcium, a messenger in the chemotaxic process. The effects of MC-LA, MC-YR and MC-LR in human neutrophils and mice had the same pattern of response. The analyses of cell viability, DNA fragmentation, mitochondrial membrane depolarization of and release of intracellular ROS were evaluated by the technique of FACS. Extracellular ROS content was measured by lucigenin-amplified chemiluminescence, and cytokines were determined by ELISA. We found that these MCs increased interleukin-8 (IL-8), cytokine-induced neutrophil chemoattractant-2αβ (CINC-2αβ) and extracellular ROS levels in human and rat neutrophils. In conclusion, our results showed that MCs act on specific pathways of neutrophil recruitment, indicating their potential effect on neutrophils activation. This process can significantly contribute to the pathogenesis of hepatic damage due to generation of ROS by neutrophils as well as act on hepatocytes under such conditions and potentially increase injury processes induced by MCs.

A capacidade de preservar a viabilidade do sêmen pelo emprego das biotecnologias, como refrigeração e criopreservação, oferece muitas vantagens a equideocultura. O estresse oxidativo é um dos principais entraves para estas biotecnicas e advêm do ataque de distintas espécies reativas de oxigênio (EROs). Assim, o objetivo do presente estudo foi verificar o impacto das diferentes EROs sobre os espermatozoides com e sem plasma seminal. Foram colhidos ejaculados de 13 garanhões Mangalarga Marchador de fertilidade conhecida. Os ejaculados foram divididos em duas frações (A e B). As frações foram centrifugadas (2200g/10min) e o pellet com os espermatozoides foi ressuspendido em solução salina fisiológica ou plasma seminal de modo que as duas frações apresentassem a mesma concentração final (sptz/mL). Ambas as frações (A e B) tiveram 4 alíquotas de 400µL retiradas e submetidas a 3 sistemas distintos de produção de EROs (xantina + xantina oxidase que produz anion superóxido; peróxido de hidrogênio; ferro + vitamina C que produz radical hidroxil) e malondialdeído. Após a incubação, as amostras A e B foram avaliadas de acordo com os testes funcionais (coloração de eosina-nigrosina para membranas, fast-green/rosa bengala para acrossomos, coloração 3-3'diaminobenzidina para atividade mitocondrial e SCSA™ para susceptibilidade a denaturação de cromatina) e avaliação do índice de peroxidação lipídica (TBARs).Todas as análises estatísticas foram realizadas pelo programa SAS System for Windows. A susceptibilidade do espermatozoide equino ao estresse oxidativo é variável de acordo com a espécie reativa de oxigênio empregada na incubação; as espécies mais deletérias no presente experimento foram a malondialdeído e principalmente o radical hidroxil o que já era esperado. Houve um efeito benéfico quanto a manutenção das funções celulares e status oxidativo em amostras cujo plasma seminal foi preservado. A proteção atribuída ao plasma seminal influenciou parâmetros como atividade mitocondrial, susceptibilidade a denaturação acida da cromatina e índice de peroxidação lipídica. O plasma seminal exerceu mais pronunciada proteção aos eventos desencadeados pela malondialdeído / Biotechnologies such as chilling and cryopreservation of equine semen have been largely used in horse breeding due to its importance on storage and propagation of genetic material. The oxidative stress is one of the main causes of poor results related to the fertility of processed stallion sperm. Oxidative injuries are mainly due to the attack of different reactive oxygen species (ROS). Thus, the aim of the present study was to evaluate the impact of distinct ROS attack on equine spermatozoa with and without seminal plasma. The collection of one ejaculate of thirteen Mangalarga fertile stallions were performed. Ejaculates were divided in two aliquots (A and B). These aliquots were centrifuged (600G /10 min) and pellets were resuspended in saline solution (0,9%) or seminal plasma certifying that each sample had the same concentration (sptz/mL). Both aliquots (A and B) were divided in four samples of 400 µL each and incubated with four ROS generating systems (superoxide anion; hydrogen peroxide; hydroxil radical and malondialdehyde). After the 30 incubation, all the samples were evaluated for membrane integrity (eosin-nigrosin stain), acrosome integrity (simple stain of fast-green/ Bengal rose), 3-3'diaminbenzidine for mitochondrial activity and SCSA™ for the chromatin suceptibility to the acid denaturation. Lipid peroxidation was accessed by TBARs assay. Statistical analysis was performed using SAS System for Windows. The susceptibility of equine sperm to the oxidative stress is different in each ROS generation system; but the most deleterious ROS were hydroxyl radical and malondialdehyde. A positive effect of seminal plasma on the sperm viability was observed which may be linked to its protective role. The protection occurred in mitochondria, chromatin and against lipid peroxidation. Higher beneficial effect of seminal plasma was observed in samples treated with malondialdehyde

Cardiomyopathies and arrhythmias are major causes of death in untreated hereditary haemochromatosis, acute iron poisoning and during secondary iron overload resulting from repeated blood transfusions in β-thalassaemia. Iron overload cardiomyopathies are associated with systolic and diastolic dysfunction, suggesting that Ca2+ homeostasis is impaired. However, the cellular mechanisms of these dysfunctions are unknown. The data presented in this thesis establishes for the first time iron effects on cardiomyocyte Ca2+ handling, as well as the potential cellular substrates responsible for this impairment during iron overload. Exposure of isolated rat ventricular cardiomyocytes to 200μM iron led to biphasic changes in systolic Ca2+ release. Phase 1: an initial reduction of systolic Ca2+ release followed by; Phase 2: increased Ca2+ release with arrhythmogenic spontaneous Ca2+ release, cell contracture and cell death. There is evidence that Fe2+ enters cardiomyocytes via L-type Ca2+ channels (LTCC) and reduces the Ca2+ trigger. The close apposition of LTCCs to cardiac ryanodine receptors (RyR2) suggests RyR2 may be a first target. Indeed RyR2 activity was drastically reduced on exposure to nanomolar [Fe2+] in single channel studies. Together with evidence that Fe2+ may reduce the Ca2+ trigger from LTCC, this is consistent with iron reducing sarcoplasmic reticulum (SR) Ca2+ release during Phase 1. In Phase 2, the presence of spontaneous Ca2+ release events is consistent with SR Ca2+ overload. Indeed, in single rat ventricular cardiomyocytes SR Ca2+ content was found to be increased by 27% during Phase 2. The cellular substrates responsible for this increased SR Ca2+ content were 2-fold: 1) through reduced extrusion via both the Na+ Ca2+ Exchanger (NCX) and Plasmalemmal Ca2+ ATPase (PMCA) and 2) through increased resequestration via the SR Ca2+ ATPase. Iron catalyses the production of reactive oxygen species (ROS) during the Fenton reaction. To investigate whether iron effects might be due to ROS, I used the cell permeant ROS scavenger Tempol. Tempol attenuated Phase 2 effects but Phase 1 effects were not affected. This is consistent with the hypothesis that Phase 1 effects were due to direct effects of Fe2+ affecting LTCC trigger and RyR2 function. The attenuation of Phase 2 effects suggests that ROS damage to key Ca2+ handling mechanisms, such as NCX and PMCA might account for a reduced Ca2+ extrusion and subsequent SR Ca2+ overload.

Bicarbonato é uma importante espécie química para os seres vivos, sendo o principal tampão celular, alem de apresentar uma negligenciada atividade redox. Isquemia é um evento no qual existe inibição do aporte de nutrientes e oxigênio, sendo a reperfusão o retorno do fluxo de nutrientes e oxigênio, que é acompanhada por alta produção de radicais livres e morte celular. Nessa tese estudamos o efeito da presença de bicarbonato durante a isquemia-reperfusão. Em nosso modelo nós mantivemos o pH constante e modulamos a quantidade de bicarbonato enquanto células, órgãos e animais foram submetidos a isquemia-reperfusão. Utilizamos condições sem a presença de bicarbonato, a concentração basal sanguínea e uma concentração mais alta simulando o acúmulo de bicarbonato em condições isquêmicas. Nesses diversos modelos mostramos que a presença de bicarbonato aumenta o dano provocado por isquemia-reperfusão e provoca um aumento do acúmulo de proteínas oxidadas. A presença do bicarbonato não modifica a respiração, produção de espécies reativas de oxigênio, ou a morfologia mitocondrial, também não detectamos mudança na atividade do proteassoma e nos indicadores de autofagia geral. Entretanto detectamos um acúmulo de marcadores autofágicos na fração mitocondrial indicando inibição da mitofagia. Essa inibição foi confirmada ao detectarmos o acúmulo de uma proteína degradada especificamente por mitofagia enquanto não houve mudança em outra degradada pelo proteassoma. Além disso, ao inibirmos farmacologicamente a autofagia, reproduzimos o fenótipo causado pelo bicarbonato mesmo na sua ausência. Em conclusão, a presença de bicarbonato é deletéria em condições de isquemia/reperfusão devido a inibição da mitofagia / Bicarbonate is an important molecule in all living being, acting as the main cellular buffer. However, its biological and redox activity has been mostly neglected to date. Ischemia is an event in which an inhibition of nutrient availablity and oxygen flow occurs, while reperfusion is the return of nutrients and oxygen, accompanied of a burst of reactive oxygen species production and cell death. Here, we studied the effects of bicarbonate during cardiac ischemia-reperfusion. In our model, we kept the pH stable and changed the concentration of the bicarbonate. We then subjected cells, organs and animals to ischemia-reperfusion under conditions where there was no presence, basal blood concentration or a higher concentration of bicarbonate. In these diverse models, we found that the presence of bicarbonate increased damage after a ischemia-reperfusion, and promoted the accumulation of oxidized proteins. Bicarbonate did not change respiration, production of reactive oxygen species or the morphology of the mitochondria. There were also no changes in proteasome activity and in global autophagy markers, although there was an accumulation of mitophagy markers. We also found that mitophagy was responsible for the increased damage observed, since pharmacological inhibiting of autophagy abolished the increased damage caused by the presence of bicarbonate. In conclusion the presence of bicarbonate is deleterious in ischemia-reperfusion due mitophagy inhibition

The carbonate radical anion (CO3•-, CR) is an important reactive oxygen species produced in vivo by one-electron oxidation of CO2 or bicarbonate, constituents of the major physiological buffer. It was demonstrated for the first time by using an HPLC-based analysis of low-molecular products of DNA damage that CRs react with DNA 2΄-deoxyribose by the hydrogen abstraction mechanism. CRs exhibit a ~ 800-fold preference for one-electron oxidation of guanine over hydrogen abstraction from DNA sugar, in sharp contrast with •OH. CRs also have, as compared to •OH, an increased preference for the H1΄ abstraction, which is the most thermodynamically favorable due to the highest stability of the respective deoxyribosyl radical but kinetically the slowest due to low solvent accessibility, by the expense of the decreased preference for the H5΄ abstraction. All these findings are in agreement with the characteristics of CR as a potent oxidant and selective hydrogen abstractor.

8-oxo-7,8-dihydroguanine (8oxoG) is one of the most important base lesions formed during oxidative damage of DNA. The aim of the present research was to investigate the effects of DNA concentration, G content, and the nature of oxidizing species on the kinetics of 8oxoG in model DNA solutions by using HPLC. The experimentally obtained yields of 8oxoG were typically in the range of 2-2.5% of total concentration of guanine. The ratios of the rate constant of hole diffusion in DNA to the rate constant of conversion of the hole into 8oxoG (kd/kr) were calculated from the experimental data using the diffusion model of charge transfer in DNA to be in the range of 200-300, in agreement with previously reported kd/kr ratios in the duplex DNA oligonucleotides (GGA)n or (GGTT)n. Our current diffusion model cannot satisfactorily explain the absence of the G content dependence of the 8oxoG yields, which indicates that a more advanced model is required.

Methamphetamine (METH) is a highly addictive substance that is highly prevalent in today’s society, with over 1 in 20 adults over 26 having taken it at least once. While it is known that METH, a common psychostimulant, acts on both the mesolimbic dopamine (DA) and nigrostriatal DA systems by affecting proteins involved in DA reuptake and vesicular packaging, the specific mechanism of what is known as METH neurotoxicity remains obscure, but has been shown to involve oxidative stress. Studies have shown that reactive oxygen species act on the same proteins that METH affects. Oxidative species have also been known to catalyze the formation of melanins in dopaminergic cells. We explore this link more fully here. In an in vitro system, oxidative species (including Fe3+, an inorganic catalyst for oxidative stress), enhance the rate of melanization of DA. Methamphetamine increased oxidative stress in an in vivo model. Additionally, METH enhanced phasic (stimulated) DA release and caused an electrically-independent efflux of DA. Lidocaine abolished phasic DA release, but did not affect METH-induced DA efflux, indicating action-potential dependent and independent mechanisms behind METH’s effects. The sigma-1 receptor antagonist BD 1063 significantly attenuated METH’s effect on DA release. Depletion of intracellular calcium (Ca2+) reserves also attenuated METH-enhancement of DA release. We investigated the role of oxidative species in METH-induced DA efflux. Reduced glutathione (the substrate for glutathione peroxidase) and 4-hydroxy-TEMPOL (a superoxide dismutase mimetic) blocked METH’s effect on DA release, suggesting that a reactive oxygen species (ROS), most likely superoxide, is necessary for METH-induced DA efflux. Finally, oxidative stress as well as acute METH impairs the vesicular monoamine transporter 2 (VMAT2) by S-glutathionylation modification of Cys-488, highlighting VMAT2 as a likely regulator of METH’s effects on electrically independent DA release. These findings help outline a model in which METH induces DA release in the NAc through a signaling cascade involving the sigma receptor and ROS signaling molecules.