A hipertensão acomete aproximadamente 1 bilhão de pessoas em todo o mundo, no entanto, a sua etiologia exata é em muitos casos indefinida e o mecanismo de ação molecular não foi elucidado completamente. O óxido nítrico ( ) é liberado de forma contínua nos vasos de resistência, contribuindo para o controle fisiológico da pressão arterial, sendo que a redução em sua biodisponibilidade está correlacionada com o desenvolvimento da hipertensão. Durante muito tempo, acreditava-se que o nitrato e o nitrito eram apenas metabólitos inertes da via do e somente em 1994 foi demonstrado que o nitrito, derivado do nitrato, era um substrato para a produção do no estômago, de modo que a administração de nitrito de sódio exerce efeitos anti-hipertensivos. De fato, há diversas evidências demonstrando os efeitos do nitrito sobre a pressão arterial, porém, não há um consenso sobre o seu mecanismo de ação. Trabalhos recentes sugerem que os efeitos antihipertensivo do nitrito se baseiam na geração de S-nitrosotióis no estômago e na capacidade destas moléculas em nitrosar proteínas da parede vascular, alterando a sua função. Há diversas proteínas da parede vascular com função crítica no controle do tônus vascular que podem ser alvos para a nitrosação. Neste trabalho, optamos por avaliar o processo de nitrosação na sinalização do receptor -adrenérgico em ratos hipertensos e normotensos tratados com nitrito de sódio ou veículo pela via oral por 7 dias. Também foi realizada a incubação de anéis de aorta com nitrito de sódio ou S-nitrosoglutationa na presença ou ausência de ascorbato. O tratamento com nitrito de sódio oral por 7 dias foi responsável por reduzir a pressão arterial sistólica dos animais hipertensos e aumentar as quantidades de nitrito e espécies nitrosadas no plasma. Também foi observado uma redução na reatividade aórtica à fenilefrina em animais tratados com nitrito, sendo que este efeito é abolido na presença de ascorbato. A incubação de anéis de aorta com nitrito de sódio não demonstrou causar efeitos sobre a reatividade vascular, porém, o pré-tratamento com S-nitrosoglutationa seguido da sua remoção foi responsável por reduzir a reatividade aórtica à fenilefrina e aumentar a quantidade de proteínas nitrosadas na aorta. Estes efeitos também foram abolidos pela incubação com ascorbato. Estes resultados nos sugerem que os S-nitrosotióis, e não o nitrito, exercem efeito anti-hipertensivo através da redução da reatividade vascular na aorta, modulando a sinalização do receptor -adrenérgico. Estes efeitos aparentam se basear na nitrosação de proteínas, visto que a incubação com ascorbato é responsável por abolir estes efeitos e há um aumento na quantidade de proteínas nitrosadas na aorta após a pré-incubação com S-nitrosoglutationa / Hypertension affects approximately 1 billion people worldwide, however, its exact etiology is in many cases indefinite and the mechanism of molecular action has not been elucidated completely. Nitric oxide ( ) is released continuously in resistance vessels, contributing to the physiological control of blood pressure, and the reduction in its bioavailability is correlated with the development of hypertension. For a long time, it was believed that nitrate and nitrite were only inert metabolites of the pathway, and it was only in 1994 that nitrite derived from nitrate was shown to be a substrate for the production of in the stomach, so that administration of sodium nitrite exerts antihypertensive effects. In fact, there is a lot of evidence demonstrating the effects of nitrite on blood pressure, but there is no consensus on its mechanism of action. Recent work has suggested that the antihypertensive effects of nitrite are based on the generation of S-nitrosothiols in the stomach and on the ability of these molecules to nitrosate proteins in the vascular wall, altering their function. There are several vascular wall proteins with critical function in vascular tone control that may be targets for nitrosation. In this work, we chose to evaluate the process of nitrosation in -adrenergic receptor signaling in hypertensive and normotensive rats treated with sodium nitrite or vehicle orally for 7 days. Aortic rings were also incubated with sodium nitrite or S-nitrosoglutathione in the presence or absence of ascorbate. Treatment with oral sodium nitrite for 7 days was responsible for reducing the systolic blood pressure of hypertensive animals and increasing the amounts of nitrite and nitrosated species in the plasma. A reduction in aortic reactivity to phenylephrine has also been observed in nitrite treated animals, and this effect is abolished in the presence of ascorbate. The incubation of aortic rings with sodium nitrite was not shown to have an effect on vascular reactivity, however, pre-treatment with S-nitrosoglutathione followed by its removal was responsible for reducing aortic reactivity to phenylephrine and increasing the amount of nitrosated proteins in the aorta. These effects were also abolished by incubation with ascorbate. These results suggest that S-nitrosothiols, not nitrite, exert an antihypertensive effect by reducing vascular reactivity in the aorta, modulating -adrenergic receptor signaling. These effects appear to be based on protein nitrosation, since incubation with ascorbate is responsible for abolishing these effects and there is an increase in the amount of nitrosated proteins in the aorta after preincubation with S-nitrosoglutathione

Nitrosamines have garnered increasing attention from researchers and policy makers in recent years due to potential human health implications associated with their unintentional formation in water and wastewater treatment facilities. This work addresses a crucial nitrosamine formation pathway concerning the catalysis of amine nitrosation by activated carbon materials whose use is widespread in municipal and industrial systems. Experimental results show that this catalysis is highly pH dependent, with maximum formation achieved near the pKa value for each of the secondary amines tested. This result suggests that the overall formation potential is governed by individual amine properties and their interactions with carbon surfaces, rather than solely nitrite speciation as previously reported. Formation of the most commonly studied nitrosamine, N-nitrosodimethylamine, was shown to be highly dependent on initial dimethylamine (DMA) solution concentration, with yields of approximately 0.11% of the spiked secondary amine at pH 7.5. Morpholine and dibutylamine, larger and bulkier secondary amines, formed their corresponding nitrosamines at higher yields than DMA (0.21% and 1.69%, respectively). Additionally, select tertiary amines were shown to be capable of undergoing nitrosation on the same order of magnitude as the secondary amines under neutral conditions in the presence of activated carbons. The magnitude of these results indicates that greater attention should be paid to this previously overlooked mechanism for nitrosamine formation.

Dietary nitrite intake has been implicated in numerous gastrointestinal cancers in humans due to the formation of a group of carcinogens called N-nitroso compounds. The need to estimate their intake is vital in establishing at risk population and to monitor and perhaps one day manage their dietary intake. This is the first study to estimate nitrate and nitrite in selected vegetables, cured and fresh meat in Australian food supply using ion-paired reversed-phased HPLC. Nitrite content in meat products analysed ranged from 0 to 83.9 mg/kg in medallion beef and Frankfurt, respectively; nitrate content ranged from 18.7 mg/kg in minced beef to 142.5 mg/kg in salami. The nitrite content was below the maximum limit set by the Food Standards Australia and New Zealand. Nitrate content in selected vegetables ranged from 123 to 4850 mg/kg in Iceberg lettuce and English spinach, respectively; only minimal nitrite at 20 mg/kg was present in Gai choy, which was most likely due to bacterial contamination during storage. Based on the food consumption pattern of Australians, the dietary nitrite and nitrate intake from bacon were 1.51 and 3.42 mg per capita per day, which was below the Adequate Daily Intake set by the European Union Scientific Committee for food in 1995. Taking into considerations of oral nitrate reduction to nitrite and the endogenous nitrate formation, the upper extreme of dietary nitrite and nitrate intake in Australians were 44 and 2.4 times over the ADI, respectively. However, this does not take into effect of other dietary promoters and inhibitors. Eighteen healthy human volunteers were put on a low nitrate, nitrite and antioxidant diet for three days during which they were fed one serving of cured meat with and without 500 mg of vitamin C. Using GC-MS, N-nitrosodimethylamine was not detected in the urine before or after vitamin C supplementation, suggested that a diet low on nitrate and nitrite cannot produce NDMA and thus may reduce the risk of developing gastrointestinal cancers. Different extraction methods and combination of herbs and spices were demonstrated in vitro to show inhibition against B. cereus, Escherichia coli, Listeria monocytogenes, Salmonella Enteritidis and Staphylococcus aureus. In addition, autoclaved turmeric powder at 0.3 % (w/v), hot water extracted turmeric with ginger at 0.5 % and rosemary at 1.0 % showed growth inhibition against Clostridium sporogenes, which was used as a surrogate for Clostridium botulinum. The use of these combinations of herbs and spices may replace or at least reduce the use of nitrite as a preservative in cured meat products to prevent botulism and reduce dietary nitrite intake.

Dietary nitrite intake has been implicated in numerous gastrointestinal cancers in humans due to the formation of a group of carcinogens called N-nitroso compounds. The need to estimate their intake is vital in establishing at risk population and to monitor and perhaps one day manage their dietary intake. This is the first study to estimate nitrate and nitrite in selected vegetables, cured and fresh meat in Australian food supply using ion-paired reversed-phased HPLC. Nitrite content in meat products analysed ranged from 0 to 83.9 mg/kg in medallion beef and Frankfurt, respectively; nitrate content ranged from 18.7 mg/kg in minced beef to 142.5 mg/kg in salami. The nitrite content was below the maximum limit set by the Food Standards Australia and New Zealand. Nitrate content in selected vegetables ranged from 123 to 4850 mg/kg in Iceberg lettuce and English spinach, respectively; only minimal nitrite at 20 mg/kg was present in Gai choy, which was most likely due to bacterial contamination during storage. Based on the food consumption pattern of Australians, the dietary nitrite and nitrate intake from bacon were 1.51 and 3.42 mg per capita per day, which was below the Adequate Daily Intake set by the European Union Scientific Committee for food in 1995. Taking into considerations of oral nitrate reduction to nitrite and the endogenous nitrate formation, the upper extreme of dietary nitrite and nitrate intake in Australians were 44 and 2.4 times over the ADI, respectively. However, this does not take into effect of other dietary promoters and inhibitors. Eighteen healthy human volunteers were put on a low nitrate, nitrite and antioxidant diet for three days during which they were fed one serving of cured meat with and without 500 mg of vitamin C. Using GC-MS, N-nitrosodimethylamine was not detected in the urine before or after vitamin C supplementation, suggested that a diet low on nitrate and nitrite cannot produce NDMA and thus may reduce the risk of developing gastrointestinal cancers. Different extraction methods and combination of herbs and spices were demonstrated in vitro to show inhibition against B. cereus, Escherichia coli, Listeria monocytogenes, Salmonella Enteritidis and Staphylococcus aureus. In addition, autoclaved turmeric powder at 0.3 % (w/v), hot water extracted turmeric with ginger at 0.5 % and rosemary at 1.0 % showed growth inhibition against Clostridium sporogenes, which was used as a surrogate for Clostridium botulinum. The use of these combinations of herbs and spices may replace or at least reduce the use of nitrite as a preservative in cured meat products to prevent botulism and reduce dietary nitrite intake.

Cysteamine (CA), which is an aminothiol drug medically known as Cystagon® was studied in this thesis. Cysteamine was reacted with a binary toxin called peroxynitrite (PN) which is assembled spontaneously whenever nitric oxide and superoxide are produced together and the decomposition of peroxyinitrite was monitored. PN was able to nitrosate CA in highly acidic medium and excess CA to form S-nitrosocysteamine (CANO) in a 1:1 with the formation of one mole of CANO from one mole of ONOOH. In excess oxidant (PN) the following 1:2 stoichiometric ratio was obtained; ONOO- + 2CA → CA-CA + NO2- + H2O . In alkali medium the oxidation of CA went through a series of stages from sulfenic acid, sulfinic acid and then sulfonic acid which was followed by the cleavage of the C-S bond to form a reducing sulfur leaving group, which is easily oxidized to sulfate. The nitrosation reaction was first order in peroxynitrite, thus implicating it as a nitrosating agent in highly acidic pH conditions. Acid catalyzes nitrosation reaction, whitst nitrate catalyzed and increased the amount of CANO product, This means that the nitrosonium cation, NO+ which is produced from the protonation of nitrous acid(in situ) as also contributing to the nitrosation of CA species in highly acidic environments. The acid catalysis at constant peroxynitrite concentrations suggests that the protonated peroxynitrous acid nitrosates at a much higher rate than the peroxynitrite and peroxynitrous acid. Bimolecular rate constants for the nitrosation of CA, was deduced to be 10.23 M-1 s-1. A linear correlation was obtained between the initial rate constants and the pH. The oxidation of CA was modeled by a simple reaction scheme containing 12 reactions.

Neste trabalho foram investigados os mecanismos e o perfil cinético de processos nitrosativos do ponto de vista da nitrosação do indicador 4,5-diamino fluoresceina (DAF2) em células do tipo RAW 264.7. Também foi investigado o papel que ferro lábil (LIP) exerce em tais processos. O estudo cinético mostrou que a nitrosação do DAF2 é dependente de superóxido intracelular e se processa por dois mecanismos distintos denominados nitrosilação oxidativa e nitrosação. Observou-se que o perfil cinético da nitrosaçao do DAF2 sofre uma transição passando de dependente para independente com relação à concentração de NO, quando a concentração de NO se aproxima de 100-110nM. Este perfil está relacionado com a dinâmica de recombinação entre NO e O2¯ que dispara todo o processo de nitrosação do DAF2. No trabalho fica claro que processos nitrosativos que ocorrem pelos mesmos mecanismos podem apresentar perfis cinéticos completamente diferentes dependendo da localização onde ocorre a recombinação entre NO e O2¯. O ponto mais interessante foi a constatação de que quelantes permeáveis à membranas biológicas estimulam a nitrosação do DAF2 intracelular. Este efeito é decorrente da remoção de LIP intracelular que, surpreendementemente, apresenta papel antinitrosativo nas condições experimentais estudadas. O papel incomum antinitrosativo apresentado por LIP é analizado do ponto de vista da reação entre LIP e ONOO¯ que tem como produto nitrito, uma espécie não nitrosante. Estes resultados podem alterar a forma como LIP é visto em processos oxidativos e nitrosativos. / In this work, we investigated the mechanisms and kinetic profiles of nitrosative processes using fluorescent indicator 4,5-diaminofluorescein (DAF2) in RAW 264.7 cells. The labile iron pool (LIP) influence in nitrosative processes was also evaluated. Intracellular DAF2 nitrosation is superoxide dependent and proceeds by two distinct mechanisms: Oxidative nitrosylation and nitrosation. The former mechanism is the most relevant under all experimental conditions tested. Interestingly, the DAF2 nitrosation rate increases linearly with NO concentration of up 100-110 nM but thereafter undergoes a sharp transition and becomes insensitive to NO. This peculiar kinetic behavior has never been reported and it is linked with NO and superoxide recombination dynamics. When NO reaches a concentration capable to outcompete superoxide dismutase for superoxide, the rate of DAF2 nitrosation becomes insensitive to NO. The most striking finding is the LIP´s influence in nitrosative processes. LIP removal by cell membrane permeable metal chelantors increases DAF2 nitrosation rate significantly, suggesting tha LIP can act as an anti-nitrosant species. This increase is probably related with LIP´s direct reaction with peroxynitrite, wich produces non-nitrosant species like nitrite. This controversial LIP´s anti-nitrosative role in cellular systems is rather interesting since it can change the way we understand it´s role in nitrosative and oxidative processes.

Neste trabalho foram investigados os mecanismos e o perfil cinético de processos nitrosativos do ponto de vista da nitrosação do indicador 4,5-diamino fluoresceina (DAF2) em células do tipo RAW 264.7. Também foi investigado o papel que ferro lábil (LIP) exerce em tais processos. O estudo cinético mostrou que a nitrosação do DAF2 é dependente de superóxido intracelular e se processa por dois mecanismos distintos denominados nitrosilação oxidativa e nitrosação. Observou-se que o perfil cinético da nitrosaçao do DAF2 sofre uma transição passando de dependente para independente com relação à concentração de NO, quando a concentração de NO se aproxima de 100-110nM. Este perfil está relacionado com a dinâmica de recombinação entre NO e O2¯ que dispara todo o processo de nitrosação do DAF2. No trabalho fica claro que processos nitrosativos que ocorrem pelos mesmos mecanismos podem apresentar perfis cinéticos completamente diferentes dependendo da localização onde ocorre a recombinação entre NO e O2¯. O ponto mais interessante foi a constatação de que quelantes permeáveis à membranas biológicas estimulam a nitrosação do DAF2 intracelular. Este efeito é decorrente da remoção de LIP intracelular que, surpreendementemente, apresenta papel antinitrosativo nas condições experimentais estudadas. O papel incomum antinitrosativo apresentado por LIP é analizado do ponto de vista da reação entre LIP e ONOO¯ que tem como produto nitrito, uma espécie não nitrosante. Estes resultados podem alterar a forma como LIP é visto em processos oxidativos e nitrosativos. / In this work, we investigated the mechanisms and kinetic profiles of nitrosative processes using fluorescent indicator 4,5-diaminofluorescein (DAF2) in RAW 264.7 cells. The labile iron pool (LIP) influence in nitrosative processes was also evaluated. Intracellular DAF2 nitrosation is superoxide dependent and proceeds by two distinct mechanisms: Oxidative nitrosylation and nitrosation. The former mechanism is the most relevant under all experimental conditions tested. Interestingly, the DAF2 nitrosation rate increases linearly with NO concentration of up 100-110 nM but thereafter undergoes a sharp transition and becomes insensitive to NO. This peculiar kinetic behavior has never been reported and it is linked with NO and superoxide recombination dynamics. When NO reaches a concentration capable to outcompete superoxide dismutase for superoxide, the rate of DAF2 nitrosation becomes insensitive to NO. The most striking finding is the LIP´s influence in nitrosative processes. LIP removal by cell membrane permeable metal chelantors increases DAF2 nitrosation rate significantly, suggesting tha LIP can act as an anti-nitrosant species. This increase is probably related with LIP´s direct reaction with peroxynitrite, wich produces non-nitrosant species like nitrite. This controversial LIP´s anti-nitrosative role in cellular systems is rather interesting since it can change the way we understand it´s role in nitrosative and oxidative processes.

[pt] A descoberta da importância do óxido nítrico na fisiologia humana expandiu a investigação dos mecanismos de interação das espécies reativas derivadas de óxidos de nitrogênio com biomoléculas. S-nitrosotióis são uma fonte conveniente de óxido nítrico para utilização in vivo. Um dos métodos largamente utilizados para S-nitrosilação de compostos contendo o grupo -SH, incluindo proteínas contendo o aminoácido cisteína, é a reação com nitrito em meio ácido. Nesse trabalho investigaram-se as espécies derivadas da reação de albumina sérica, humana e bovina, e de insulina com nitrito, através de espectrofotometria de absorção e de fluorescência. Além da modificação dos resíduos de cisteína, demonstrou-se que resíduos do aminoácido triptofano também sofrem modificação, podendo participar dos processos in vivo. A partir da comparação entre os espectros de absorção dos cromóforos formados em albumina humana e bovina, bem como em insulina, demonstrou-se que a banda de absorção no ultravioleta, descrita na literatura como característica de S-nitrosilação de cisteína, é dominada pelos resíduos de triptofano N-nitrosados. Experimentos de fluorescência confirmaram a modificação dos resíduos de triptofano, já que os espectros apresentaram redução do rendimento quântico e também deslocamento do pico característico desses resíduos. Acompanhou-se a cinética de formação de novos cromóforos, comparando as albuminas nativas e bloqueadas no resíduo de cisteína. Investigou-se a cinética de modificação dos resíduos de triptofano em pH fisiológico através de fluorescência. / [en] The discovery of the importance of nitric oxide to the human physiology expanded the investigation of mechanisms involved in the interactions of nitrogen oxide reactive species with biomolecules. S-nitrosothiols are a convenient source of nitric oxide for in vivo applications. Acid treatment with nitrite of compounds containing the SH group, including proteins containing cysteine residues, is a widely used method to synthesize S-nitrosothiols. In this work, several species derived from the nitrite acid treatment of human and serum albumins as well as insulin were investigated using optical absorption and fluorescence. It was demonstrated that, besides cysteine, tryptophan residues are modified and can participate in processes in vivo. Comparing the absorption spectra from human and bovine serum albumin with that from insulin, it was demonstrated that the ultraviolet absorption band, described in the literature as coming from Snitrosylation, was mainly due to N-nitrosation of tryptophan residues. Fluorescence experiments confirmed the modification of tryptophan residues, since the characteristic fluorescence peak exhibited a reduction and a blue shift. The kinetics of the new chromophores was followed by comparison of native and cysteine-blocked albumins. The kinetics of tryptophan modifications was investigated at the physiological pH using fluorescence.