A concentração de ferro heme é um parâmetro de qualidade da carne, daí a importância de ser avaliado em carnes processadas. O objetivo deste trabalho foi avaliar a preservação do heme na cocção e no congelamento. Foram realizados dois experimentos com cortes de coxão mole (m. semi membranosus) moído, de origem bovina e em formato de hambúrgueres. Experimento1: a carne foi preparada em forno combinado a 150, 240 e 300ºC por 3, 5 e 10 minutos com umidade no forno de 10, 60 e 100%; em forno elétrico, a 150, 200 e 280ºC, por 6, 12 e 24 minutos e em chapa elétrica (200ºC) por 45, 120 e 240 segundos. Experimento 2: a carne foi congelada de forma lenta e rápida e armazenada por três meses. Foram determinadas as concentrações de ferro heme (FeH) pelo método de Hornsey, (1956) e modificado por Carpenter e Clark (1995), e avaliados parâmetros cinéticos da ruptura do heme. Esse efeito na carne cozida em todos os equipamentos mostrou ser bastante dependente da concentração. A redução da concentração de ferro heme durante a cocção seguiu cinética de segunda ordem. A partir da concentração de ferro heme inicial e das condições de cocção, as equações obtidas permitiram calcular as concentrações finais de ferro heme após preparação do coxão mole em forno combinado, chapa elétrica e forno elétrico com erros de 3,9; 5,7 e 17,9%, respectivamente. O congelamento, tanto rápido, como lento, não alterou a concentração do FeH. Com este estudo foi possível identificar as condições de preparo mais indicadas para a preservação do FeH em coxão mole, e os resultados poderão conferir um refinamento aos dados de tabelas de composição de alimentos, bem como servir como ferramenta de trabalho para profissionais que estão à frente de Unidades de Alimentação e Nutrição. / Heme iron concentration is a meat quality parameter, hence its importance when evaluating processed meat. The aim of this work was to evaluate the preservation of heme iron during cooking and freezing. Two experiments with semi membranosus muscle hamburgers were carried out. Experiment 1 - the meat was cooked in a combined oven (with humidity levels of 10, 60 and 100%) at 150, 240 and 300ºC for 3, 5 and 10 minutes; in an electric oven at 150, 200 and 280ºC for 6, 12 and 24 minutes and on an electric fryer (200ºC) for 45, 120 and 240 seconds. Experiment 2- two meat groups (one slowly frozen and another rapidly frozen) were stored for 3 months. The concentrations of heme iron were determined by the Hornsey method (1956) and modified by Carpenter and Clark (1995), and the kinetic parameters regarding heme breakage were evaluated. This effect on meat in all equipment depended largely on the concentration. The decrease in iron concentration during cooking followed a second-order kinetics. From the initial concentrations of heme iron and cooking conditions, the equations that were obtained allowed to calculate the final concentration of heme iron, after cooking the meat in combined oven, on electric fryer and in electric oven, with an error of 3.9, 5.7 and 17.9%, respectively. Both rapid and slow freezing did not alter the concentration of heme iron. This study allowed identifying the most appropriate cooking conditions to preserve heme iron in meat, and the results may contribute to the quality of data in food composition tables and also serve as a tool for professionals in charge of Food and Nutrition Units.

A long standing global scientific challenge has been the activation of O2 at a single metal center, and use of the subsequent metal-based oxidant for a variety of difficult chemical transformations. Towards this end, computational and synthetic methods have been utilized in an approach to develop model compounds capable of this type of chemistry, and to better understand the electronic and mechanistic properties of the observed catalytic reactivity. We have developed a first generation catalyst that has been shown to be fully functional in utilizing α-keto acids for the catalytic activation of O2 and oxidation of organic substrates in a highly conserved manner. This reactivity takes place at room temperature and standard pressure, and resembles the type of chemistry performed by mononuclear non-heme enzymes, which inspired the design of the catalyst. However, these solution-phase reactions do not benefit from the controlled environment provided by a protein active site, and solution studies and DFT simulations demonstrate an isomeric family of reactive species that ultimately deactivate via a dimerization pathway. A second generation catalyst, which incorporates ligand aromatic functionality, has been developed. This complex has been shown to catalytically oxide methanol to formaldehyde in the presence of α-ketoglutarate using O2. The aromatic group provides a synthetic platform, allowing a variety of substituents geared toward increasing complex solubility and the tuning of the redox properties of the metal center. Additionally, the ligand has been functionalized to allow for the immobilization of the catalyst using an azido-functionalized solid support, by means of 'click' chemistry. A procedure for the immobilization of the catalyst has been developed that sets the stage for the preparation of a material that will diminish dimerization and inactivation. Additional insights into potential reaction pathways of the first generation catalyst have been obtained from DFT studies. These simulations have provided energetic comparisons of proposed intermediates and set the stage for future computational and spectroscopic studies. This synergistic approach will not only allow for detailed electronic and mechanistic descriptions of the intimate mechanism, but will be used in the development of next generation catalysts that that can be tuned for desired reactivity properties.

A concentração de ferro heme é um parâmetro de qualidade da carne, daí a importância de ser avaliado em carnes processadas. O objetivo deste trabalho foi avaliar a preservação do heme na cocção e no congelamento. Foram realizados dois experimentos com cortes de coxão mole (m. semi membranosus) moído, de origem bovina e em formato de hambúrgueres. Experimento1: a carne foi preparada em forno combinado a 150, 240 e 300ºC por 3, 5 e 10 minutos com umidade no forno de 10, 60 e 100%; em forno elétrico, a 150, 200 e 280ºC, por 6, 12 e 24 minutos e em chapa elétrica (200ºC) por 45, 120 e 240 segundos. Experimento 2: a carne foi congelada de forma lenta e rápida e armazenada por três meses. Foram determinadas as concentrações de ferro heme (FeH) pelo método de Hornsey, (1956) e modificado por Carpenter e Clark (1995), e avaliados parâmetros cinéticos da ruptura do heme. Esse efeito na carne cozida em todos os equipamentos mostrou ser bastante dependente da concentração. A redução da concentração de ferro heme durante a cocção seguiu cinética de segunda ordem. A partir da concentração de ferro heme inicial e das condições de cocção, as equações obtidas permitiram calcular as concentrações finais de ferro heme após preparação do coxão mole em forno combinado, chapa elétrica e forno elétrico com erros de 3,9; 5,7 e 17,9%, respectivamente. O congelamento, tanto rápido, como lento, não alterou a concentração do FeH. Com este estudo foi possível identificar as condições de preparo mais indicadas para a preservação do FeH em coxão mole, e os resultados poderão conferir um refinamento aos dados de tabelas de composição de alimentos, bem como servir como ferramenta de trabalho para profissionais que estão à frente de Unidades de Alimentação e Nutrição. / Heme iron concentration is a meat quality parameter, hence its importance when evaluating processed meat. The aim of this work was to evaluate the preservation of heme iron during cooking and freezing. Two experiments with semi membranosus muscle hamburgers were carried out. Experiment 1 - the meat was cooked in a combined oven (with humidity levels of 10, 60 and 100%) at 150, 240 and 300ºC for 3, 5 and 10 minutes; in an electric oven at 150, 200 and 280ºC for 6, 12 and 24 minutes and on an electric fryer (200ºC) for 45, 120 and 240 seconds. Experiment 2- two meat groups (one slowly frozen and another rapidly frozen) were stored for 3 months. The concentrations of heme iron were determined by the Hornsey method (1956) and modified by Carpenter and Clark (1995), and the kinetic parameters regarding heme breakage were evaluated. This effect on meat in all equipment depended largely on the concentration. The decrease in iron concentration during cooking followed a second-order kinetics. From the initial concentrations of heme iron and cooking conditions, the equations that were obtained allowed to calculate the final concentration of heme iron, after cooking the meat in combined oven, on electric fryer and in electric oven, with an error of 3.9, 5.7 and 17.9%, respectively. Both rapid and slow freezing did not alter the concentration of heme iron. This study allowed identifying the most appropriate cooking conditions to preserve heme iron in meat, and the results may contribute to the quality of data in food composition tables and also serve as a tool for professionals in charge of Food and Nutrition Units.

Variable-temperature stopped-flow (VT-SF) electronic spectroscopy (-85 to -50°C) was utilized to study the reactivity properties of a family of synthetic mononuclear and binuclear non-heme iron enzyme active site analogs. This technique was used to investigate the mechanisms of interactions of two diiron complexes, the diferrous [FeII2(H2Hbamb)2(NMI)2] and the mixed valent [FeII,FeIII(H2Hbamb)2]+, with either oxygen-atom donor (OAD) molecules or the mechanistic probe peroxide, 2-methyl-1-phenylprop-2-yl hydroperoxide (MPPH), and substrates containing weak C-H and O-H bonds. Single turnover studies with 9,10-dihydroanthracene (9,10-DHA) and the deuterated analog, d4-9,10-DHA allowed for the determination of kinetic isotope effects (KIE) which show an inverse KIE and evidence of a disproportionation mechanism. Previous investigations showed the rate of catalytic oxidation of cyclohexane to cyclohexanol by [FeII2(H2Hbamb)2(NMI)2] and MPPH decreased over time. Current VT-SF data show evidence of product inhibition by means of a pre-equilibrium process that inhibits the reaction of the oxidant with the [FeII,FeII] complex. Also examined is the ability of the [FeII,FeIII(H2Hbamb)2]+ complex to catalytically oxidize phenols to phenoxyl radicals via a putative [FeIV=O] species. The reactivity properties of substituted phenols that vary in their oxidation potentials and bond dissociation energies (BDE) was investigated by VT-SF electronic spectroscopic studies to gain insight into the mechanism of oxidation by the [FeII,FeIII] complex. Mechanistic studies were also performed utilizing a mononuclear non-heme iron complex [FeII(N2O1)(CH3OH)Cl2], which can bind alpha-keto acids (e.g. alpha-ketoglutarate, benzoylformate) in a bidentate fashion. Reactivity studies utilizing O2 shows coupled decarboxylation of the alpha-keto acid with catalytic oxidation of the methanol solvent formaldehyde (285 turnovers). In non-reactive solvents, the [FeII(N2O1)(alpha-KG)] adduct complex is capable of catalytically oxidizing a variety of substrates such as 9,10-dihydroanthracene, 2,4-di-tert-butyl phenol, cyclohexene, and cyclooctane at 25°C utilizing O2 as the oxidant. Investigations to the binding of alpha-keto acids to the mononuclear iron complex in the absence of O2 by VT-SF as well as binding studies with NO are discussed. Finally, VT-SF studies were performed to probe the reaction of O2 with [FeII(N2O1)(alpha-KG)(CH3OH)] and the proposed mechanism is discussed. The relevance of these data to non-heme iron enzymes like soluble Methane Monooxygenase, Ribonucleotide Reductase, and Taurine Dioxygenase is discussed.

Chicken breast muscle was cooked to an internal temperature of 165oF by four methods: boiling, baking, sautéing and deep-frying. All cooking methods led to a decrease in formation of dialyzable iron, formed by both extraction and digestion in vitro, compared to raw muscle. After cooking most of the dialyzable iron formed results from extraction and the formation of dialyzable iron by digestion is essentially eliminated. Cooking also decreased the levels of cysteine and histidine; these losses may contribute to the loss in dialyzable iron.

Myoglobin (Mb) oxidation, and the subsequent browning, is the primary basis for consumer rejection of fresh retail beef. Considerable effort has been directed by the industry towards the development of techniques that can enhance color stability. However, the underlying mechanism of Mb oxidation has been studied extensively, but is still not entirely understood. It is known that chelation of iron and copper delays Mb oxidation and browning, but a clear role for these metals has not been established in any current Mb oxidation mechanism. The objective of the current study was to examine the possibility that iron plays a more direct role in Mb oxidation, and that metal chelators such as milk mineral (MM) and sodium tripolyphosphate can inhibit this action. MM, a colloidal calcium phosphate of large molecular weight and undetermined structure, was demonstrated to be a high-affinity iron chelator. Non-heme iron was found to stimulate Mb oxidation even in the absence of lipid, showing for the first time that the role of ferrous iron was not limited to promoting lipid oxidation, but instead has a yet-to-be determined role as a pro-oxidant factor in Mb oxidation. Ferrous iron was found to promote Mb oxidation under standard atmospheric conditions, while in high oxygen systems this effect was not seen. Addition of catalase did not affect Mb oxidation. However, in iron-containing systems, catalase significantly slowed Mb oxidation, while MM addition completely reversed the stimulatory effect of added iron. Type I radical-quenching antioxidants were found to rapidly reduce ferric iron to the ferrous form. This strong reducing ability accounted for the pro-oxidant effects of rosmarinic acid and eugenol in the lipid-free Mb model system. In raw ground beef, Type I antioxidants were highly effective at preventing Mb oxidation in the presence of lipid. Of the Type II chelators examined, only MM was able to delay Mb oxidation as well as the Type I antioxidants, possibly because it is not as susceptible to enzymatic hydrolysis.

La viande rouge est un facteur de risque du cancer colorectal, considérée comme probablement cancérigène chez l'Homme. Le lien entre la viande rouge et le cancer colorectal impliquerait la lipoperoxydation induite par le fer héminique, aboutissant à la formation d'aldéhydes cytotoxiques présents dans les eaux fécales des rats ayant consommé de l'hème, dont le 4-hydroxynonénal (HNE). Ces eaux fécales ont préférentiellement induit la mort cellulaire dans des cellules coliques murines normales plutôt que dans des cellules coliques murines mutées pour le gène APC (adenomatous polyposis coli), considérées comme prénéoplasiques. Cette résistance des cellules prénéoplasiques est associée à une activité plus élevée du Nrf2 (Nuclear factor (erythroid-derived)-like 2) par rapport aux cellules normales, mise en evidence par l'invalidation du Nrf2. Afin de valider l'importance des aldéhydes néoformés derives de la lipoperoxydation, nous avons optimisé la depletion des composes carbonyls dans les eaux fécales qui a aboli le différentiel de mortalité entre les cellules normales et prénéoplasiques. En utilisant des cellules épithéliales coliques humaines (CECH) afin de se rapprocher de la physiologie humaine, nous avons confirmé que le HNE et les eaux fécales des rats ayant consommé de l'hème ont aussi induit une mortalité plus élevée dans les CECHs normales que dans celles invalidées pour APC ; et qu'une activité de Nrf2 plus élevée est aussi observée dans ces dernières. L'autophagie, dont le niveau est plus élevé dans les CECHs invalidées pour APC, exercerait des effets protecteurs contre la toxicité du HNE et des eaux fécales en activant Nrf2. Globalement, nos résultats suggèrent que le Nrf2 et l'autophagie seraient impliqués dans la résistance des cellules prénéoplasiques suite à une exposition aux eaux fécales des rats ayant consommé de l'hème. Ce différentiel de résistance pourrait expliquer l'effet promoteur de la viande rouge sur la cancérogenèse colorectale, en établissant une sélection positive des cellules prénéoplasiques au détriment des cellules normales. / Red meat is a factor risk for colorectal cancer, considered as probably carcinogenic to humans. Red meat is associated to colorectal cancer through the oxidative properties of heme iron released in the colon. This latter is a potent catalyst for lipid peroxidation, resulting in the neoformation of deleterious aldehydes present in the fecal water of heme-fed rats, including 4-hydroxynonenal (HNE). Fecal water of heme-fed rats preferentially induced mortality in mouse normal colon epithelial cells than in those harboring mutation on APC (adenomatous polyposis coli) gene, considered as preneoplastic. This relative resistance of preneoplastic cells to fecal water of heme-fed rats was associated with their higher activity of Nrf2 (Nuclear factor (erythroid-derived 2)-like 2) compared to normal cells, as evidenced by Nrf2 invalidation. To investigate the importance of secondary aldehydes derived from lipid peroxidation, depletion of carbonyl compounds in the fecal water was optimized and this turned out to abolish the difference of mortality between normal and preneoplastic cells. By using human colon epithelial cell (HCEC) lines to approach human physiology, we confirmed that HNE and fecal water of heme-fed rats also induced higher mortality in normal HCECs compared to APC-invalidated HCECs, and that higher Nrf2 activity in APC-invalidated HCECs was also associated with such a difference. Furthermore, autophagy, found to be up-regulated in APC-invalidated HCECs, was suggested to exert protective effects against HNE and fecal water toxicity by activating Nrf2. Taken together, these findings suggest that Nrf2 and autophagy were potentially involved in the resistance of preneoplastic cells upon exposure to HNE or fecal water of heme-fed rats. This different resistance could explain the promoting effect of red meat and heme-enriched diet on colorectal cancer, by initiating positive selection of preneoplastic cells over normal cells.

Le 4-hydroxy-2-nonénal (HNE), un aldéhyde --insaturé, produit secondaire de la peroxydation des acides gras polyinsaturés en n-6 qui est cytotoxique et génotoxique in vitro. Cet aldéhyde a été largement étudié dans divers états pathologiques dans lesquels sa formation est la conséquence d’un stress oxydant associé à un état inflammatoire. Cependant, outre sa formation endogène, le HNE peut également être formé à partir des lipides alimentaires, entraînant sa présence dans les aliments mais également sa néoformation dans le tractus digestif. Ainsi, le rôle du HNE est suspecté dans la cancérogénèse colorectale induite par le fer héminique. L’effet promoteur du fer héminique, présent en grande quantité dans la viande rouge, a été démontré dans des modèles animaux, aux stades prénéoplasique et tumoral et a également été associé à la lipoperoxydation luminale. Ce travail a eu pour vocation la caractérisation du métabolisme du HNE alimentaire et l’étude de son rôle dans la cancérogénèse colorectale associée à la consommation de viande rouge, en explorant un possible effet proinflammatoire. Au préalable, nous avons démontré expérimentalement l’effet initiateur du fer héminique dans la cancérogénèse colorectale en plus de l’effet promoteur déjà validé. Concernant l’étude de métabolisme, nous avons identifié et caractérisé les métabolites du HNE présents dans l’urine suite à une exposition par voie orale chez le rat grâce à une méthode de suivi isotopique. Les voies majeures de métabolisation du HNE ont été identifiées et quantifiées. De plus, de nouveaux métabolites urinaires tel que des conjugués thiométhyles et glucuronides ont été mis en évidence pour la première fois. Les études de cancérogénèse ont porté sur les effets co-initiateurs ou promoteurs des régimes. De plus, les liens entre l’inflammation et le cancer du côlon étant souvent évoqués dans la littérature, nous avons également testé les effets du HNE sur l’inflammation colique. Au cours d’études court terme chez le rat F344, nous avons démontré l’absence d’effet du HNE sur l’inflammation et la perméabilité colique. Dans l’étude de co-initiation testant l’effet d’un régime alimentaire contenant du fer héminique ou du HNE pendant 15 jours suivi d’une initiation chimique par l’azoxyméthane, nous avons démontré pour la première fois un effet co-initiateur du fer héminique mais pas du HNE sur la cancérogénèse colorectale, au stade prénéoplasique. Dans l’étude de promotion de la cancérogénèse colorectale sur rats chimio-induits, nous avons également démontré une absence d’effet au stade prénéoplasique d’un régime riche en HNE donné pendant 100 jours après la chimio-induction. En conclusion, cette thèse apporte : (i) une description précise des métabolites urinaires du HNE absorbé par voie orale, (ii) une première démonstration expérimentale de l’effet co-initiateur du fer héminique sous forme hémine, (iii) une démonstration de l’absence d’effet du HNE seul et aux doses testées sur la cancérogénèse colorectale et l’inflammation colique. / This thesis focuses on 4-hydroxynonenal (HNE), an --unsaturated aldehyde by-product of polyunsaturated fatty acids n-6 peroxidation, which is demonstrated to be cytotoxic and genotoxic in vitro. This aldehyde has been extensively studied in various pathological conditions in which its formation is the result of oxidative stress associated to inflammation. However, in addition to its endogenous formation, the HNE can also be formed from dietary fats, leading to its presence in food but also to its new formation in the digestive tract. The role of HNE is suspected in heme iron induced colorectal carcinogenesis. Indeed, it has been experimentally shown that heme iron, found in large amounts in red meat, increases the incidence of preneoplastic lesions in animal models concomitantly with luminal lipoperoxidation. This work aims at characterizing the metabolism of dietary HNE and at studying its role in colorectal carcinogenesis associated with red meat consumption, because HNE is suspected to be one of reactive intermediates between heme iron and the development of this cancer. Suspected pro-inflammatory effects have also been tested. Beforehand, we have experimentally validated the initiating effect of heme iron in colorectal carcinogenesis in addition to its promoting effect already demonstrated. Regarding the metabolism study, we identified and characterized HNE metabolites in urine following oral exposure in rats using a stable isotope tracking method. The major routes of metabolism of HNE were identified as oxidation of carbons 1 and 9 (ALDH and P450 CYP 4A), - oxidation and mercapturic acids derivatives. In addition, new urinary metabolites such as glucuronide conjugates and thiomethyl were highlighted for the first time. Regarding carcinogenicity studies on the co-initiating or promoting effect of diet, we used doses of heme iron or HNE that are representative of a diet rich in meat or of a highly peroxidized diet. Furthermore, as the links between inflammation and colon cancer are often mentioned in the literature, we also tested the effects of HNE on colonic inflammation. First of all, during 2 short term studies of 2 and 3 weeks in F344 rats, we demonstrated the lack of effect of HNE on colonic inflammation and permeability. In the co-initiation study in which rats where fed for 15 days with a diet rich in heme iron or HNE, followed by chemical initiation azoxymethane, we demonstrated a co-initiating effect of heme iron but not of HNE on colorectal carcinogenesis. In the promotion study, we also have shown the lack of effect of a diet rich in HNE given for 100 days using a chemically-induced carcinogenesis model in rats. In conclusion, this thesis provides: (i) a precise description of urinary metabolites of HNE absorbed orally, (ii) a first experimental demonstration of the co-initating effect of heme iron in hemin form, (iii) a demonstration of the lack of effect of HNE on colorectal carcinogenesis and inflammation.

Le cancer colorectal est un problème de santé publique majeur à travers le monde. Les données épidémiologiques mettent en avant une association positive entre consommation de viande rouge et risque de cancer colorectal. En 2007, le fond mondial de recherche contre le cancer (WCRF) et l’institut américain de recherche contre le cancer (AICR) ont établi deux recommandations fortes qui sont de limiter la consommation de viande rouge à 500 g par semaine et d’éviter la consommation de charcuterie. Cependant, la viande rouge possède des qualités nutritionnelles intéressantes. De plus, les recommandations alimentaires sont très peu suivies par les populations appartenant aux catégories socio-professionnelles inférieures, qui consomment le plus de produits à base de viande et sont donc les plus à risque. Deux mécanismes semblent expliquer l’effet promoteur de la viande : la peroxydation des acides gras alimentaires et la formation de composés N-nitrosés, ces deux réactions étant catalysées par le fer héminique provenant de la viande rouge. Dans ce contexte, le projet SécuriViande a été mis en place afin de développer de nouveaux moyens de production qui permettraient, à terme, de diminuer le risque de cancer colorectal en inhibant les deux réactions catalysées par l’hème. Cette thèse a permis de valider expérimentalement l’association épidémiologique en montrant que la consommation de viande rouge et de charcuteries modèles induit la promotion de la cancérogenèse colorectale dans deux modèles animaux : le rat initié à l’azoxyméthane et la souris Min. De plus, mariner la viande bovine avec un mélange aqueux de raisin-olive est efficace pour diminuer le nombre de lésions précancéreuses chez le rat initié et les biomarqueurs associés à la cancérogenèse hème-induite dans les deux modèles. Cette thèse a également permis de proposer de nouveaux mécanismes pouvant expliquer l’effet promoteur de l’hème. Ainsi, le microbiote intestinal participe à la lipoperoxydation induite par le fer héminique. De plus la consommation d’hème, via la production d’aldéhydes issus de la lipoperoxydation, induit une augmentation de la perméabilité, de l’inflammation et de la génotoxicité au niveau de la muqueuse intestinale. En conclusion, cette thèse apporte : (i) une validation expérimentale de l’effet promoteur de la consommation de viande rouge et de charcuterie fraîches, (ii) un mode de prévention par la marinade de la viande bovine avec des extraits d’un mélange d’antioxydants raisin-olive, (iii) la mise en évidence de l’implication du microbiote dans la lipoperoxydation hème-induite et (iv) une meilleure caractérisation des conséquences de la consommation de fer héminique sur la muqueuse intestinale, via la formation d’aldéhydes. A terme, ces résultats pourraient être utilisés afin de mettre sur le marché des produits à base de viande plus sains vis-à-vis du risque de cancer colorectal, sans modifier les habitudes des consommateurs et ainsi de diminuer l’incidence du cancer colorectal. / Colorectal cancer is a real health issue worldwide. Epidemiological studies show positive association between red meat intake and colorectal cancer risk. In 2007, the World Cancer Research Fund (WCRF) and the American Institute for Cancer Research (AICR) made two strong recommendations: reduce fresh red meat intake less than 500 g per week and avoid cured meat. Nevertheless, red meat has nutritional interest and dietary recommendations are poorly followed by people in the lowest socio-professional category, who consume the most meat products and so who have the highest risk. Two mechanisms appear to explain promoting effect of meat: dietary fatty acids peroxidation and N-nitroso compounds formation, both reactions being catalyzed by heme iron from red meat. In this context, SécuriViande project was set up to develop new production ways which could lead to reduced colorectal cancer risk inhibiting the two reactions catalyzed by heme. This thesis has shown that fresh red meat and cured-meat intake induce colorectal carcinogenesis promotion in two animal models: azoxymethane induced-rats and Min mice. Marinate beef with an aqueous mixture of grape-olive is effective to reduce the number of precancer lesions in rats and biomarkers associated with heme-induced carcinogenesis in both models. This thesis also highlights new potential mechanisms of promoting effect of heme. Thus, microbiota is involved in heme-induced lipid peroxidation. Moreover, heme iron intake increases intestinal mucosa permeability, inflammation and genotoxicity, via aldehydes produced by lipoperoxidation. In conclusion, this thesis provides (i) an experimental validation of fresh red meat and cured meat promoting effect, (ii) a way to prevent this promoting effect by marinating beef with extracts of grape-olive antioxidants, (iii) the demonstration of microbiota involvement in heme-induced lipoperoxidation and (iv) a better characterization of heme iron intake consequences on intestinal mucosa via aldehydes formation. Ultimately, these findings could be used to market safer meat products, without changing consumer habits and so reduce colorectal cancer incidence.

Estudos da reatividade de espécies oxidantes e a interação destas espécies com estruturas sensíveis a oxidação e antioxidantes em condições biológicassão de grande importância no entendimento dos processos redox em alimentos e no corpo humano. A mioglobina é a ferro heme proteína majoritária do músculo esquelético de mamíferos e a sua ativação por peróxido de hidrogênio dá origem às espécies reativas de ferro heme hipervalentes,perferrilmioglobina e ferrilmioglobina, que podem induzir a condição de estresse oxidativo. A reação das espécies de ferro heme hipervalentes com constituintes do meio biológico ou alimentos como proteínas ou membranas podem tanto afetar a qualidade de produtos cárneos quanto causar danos celulares no trato gastrointestinal durante sua digestão.Pequenas moléculas tais como o NO, H2S e CO são produzidas endogenamente em sistemas biológicos e, além de desempenharem importantes funções na manutenção dometabolismo celular,podem apresentar atividade antioxidante.A presente Tese procurou investigara cinética e o mecanismo para a redução das espécies perferrilmioglobina e ferrilmioglobina pelo monóxido de carbono, reação esta que apresentaconstante de velocidade de segunda ordem de k2 =(3,3 ± 0,6) 102 mol L-1, a 25 oC, para a redução da espécie perferrilmioglobina. Posteriormente,foi investigada a cinética e mecanismo de redução da ferrilmioglobinapelo H2S levando à formação da espécie sulfomioglobina-Fe(II). A constante de segunda ordem obtida para a reação entre a espécie protonada da ferrilmioglobina e o H2Sfoide k2 = (2,5 ± 0,1) 106 L mol-1 s-1, duas ordens de magnitude superior a constante de velocidade paraa reação entre a espécie ferrilmioglobina e o íonHS-, k2 = (1,0 ± 0,7)104L mol-1 s-1a 25 oC.Para a redução da espécie ferrilmioglobina pelo H2S/HS- e a formação da espécie sulfomioglobina-Fe(II) observa-se um efeito de compensação de temperatura (?H? = (2,1 ± 0,9) kJ mol-1) o que é um fator determinante na ocorrência do processo de greening em produtos cárneos condimentados durante estocagem a baixa temperatura. A formação da espécie sulfomioglobina foi também investigada na reação de redução da ferrilmioglobina pela L-cisteína. Para esta reação foi observada dependência do mecanismo da reação com o pH. A formação da espécie sulfomioglobina foi observada para a reação conduzia em meio ácido a neutro, enquanto que para a reação em condições alcalinas, observa-se a formação majoritária da espécie oximioglobina. Areação da cisteína com a espécie protonada da ferrilmioglobina apresentou constante de segunda ordem de k2 = (5,1 ± 0,4)L mol-1 s-1, e a reação entre a cisteína diânion e a ferrilmioglobina,em meio alcalino,apresentou constante de velocidade de segunda ordem com k2 =(0,12 ± 0,01) L mol-1s-1.A diferença de reatividade e no produto da reação é um indicativo da mudança de mecanismo de transferência de elétrons seguida de adição do radical HSo, em meio ácido, para um mecanismo sequencial detransferência de elétrons do dianion da cisteína para as espécies ferrilmioglina e metamioglobina levando a formação de oximioglobina em condições alcalinas. / Studies on the reactivity and interaction of oxidant species with oxidizable sensitive structures and antioxidants in biological settings are of great importance for understanding the redox process in food and in the human body. Myoglobin is the major heme-iron protein in the mammal skeletal muscle and its activation by hydrogen peroxide generate reactive hypervalent heme-iron species, perferrylmyoglobin and ferrylmyoglobin, which may induce oxidative stress conditions. The reaction of hypervalent heme-iron species with biological medium or food constituents like proteins or membranes may affect the quality of meat products or could lead to cellular damage in the gastrointestinal tract during digestion. Small molecules like NO, H2S, and CO are produced endogenously in biological systems and, despite playing relevant function in the maintenance of cell metabolism, may present antioxidant activity. The present Thesis aimed to investigate the kinetics and mechanism for the reduction of perferrylmyoglobin and ferrylmyoglobin species by carbon monoxide, reaction that shows a second-order reaction constant with k2 = (3.3 ± 0.6) 102L mol-1 s-1at 25 oC for the reduction of the perferrylmyoglobin species. Furthermore, the kinetics and mechanism for the reduction of the ferrylmyoglobin by H2S leading to the formation of the sulfmyoglobin-Fe(II) species has been investigated. The obtained second-order rate constant for the reaction between the protonated ferrylmyoglobin species and H2S was k2 = (2.5 ± 0.1) 106 L mol-1 s-1, two-orders of magnitude higher than the second-order rate constant for the reaction between the ferrylmyoglobin species and the HS- ion, k2 = (1.0 ± 0.7) 104L mol-1 s-1at 25 oC. For the ferrylmyoglobin species reduction by H2S/HS- and the formation of sulfmyoglobin-Fe(II) it is observed an temperature compensation effect (?H? = (2.1 ± 0.9) kJ mol-1) which is the determination factor for the occurrence of the greening process in condiment meat products during storage at low temperature. The formation of the sulfmyoglobin species has been further investigated during the reduction reaction of ferrylmyoglobin by L-cysteine. For this reaction it was observed a dependence of the reaction mechanism on the pH. The formation of sulfmyoglobin was observed for the reaction conducted in acidic and neutral medium, meanwhile for the reaction in alkaline conditions, it is mainly observed the formation of oxymyoglobin. The reaction between cysteine and the protonated ferrylmyoglobin species shown second-order rate constant with k2 = (5.1 ± 0.4) L mol-1 s-1, and the reaction between the cysteine dianion and ferrylmyoglobin, in alkaline medium, shows second order rate constant with k2 = (0.12 ± 0.01) L mol-1s-1.The difference in reactivity and in the reaction product is an indicative of change of the electron transfer-radical addition mechanism to a sequential electron transfer mechanism from the cysteine dianion to the ferrylmyoglobin and metmyoglobin species leading to the formation of oxymyoglobin under alkaline conditions.