Parenteral administration of vitamins A, D and E on the oxidative metabolism and function of polymorphonuclear leukocytes in swine

The weaning period of piglets is characterized by physiological alterations, such as decreased weight gain, increased reactive oxygen species (ROS) and increased serum cortisol levels with possible effects on the immune response. The effect of parenteral administration of vitamins A, D and E on production performance, oxidative metabolism, and the function of polymorphonuclear leukocytes (PMNLs) was assessed in piglets during the weaning period. The sample was comprised of 20 male piglets that were given an injectable ADE vitamin combination (135,000 IU vitamin A, 40,000 IU vitamin D and 40mg vitamin E/animal) at 20 and 40 days of age. Weight gain, concentration of reduced glutathione (GSH), malondialdehyde (MDA), superoxide dismutase (SOD) and the microbicidal and phagocytic activity of PMNLs were assessed. No difference was observed in the average piglet weight during the study; however, a greater percentage of weight gain was observed after weaning in the treated group. The concentrations of GSH and SOD did not differ between groups, although lipid peroxidation was greater in the control group at 60 days of age. The investigated variables of oxidative metabolism were correlated as follows: -0.41 for GSH and MDA, -0.54 for GSH and SOD and 0.34 for MDA and SOD. The intensity of intracellular ROS production, the percentage of ROS-producing PMNLs and the intensity of phagocytosis by PMNLs did not differ between treatment groups. Administration of the injectable ADE combination improved the percentage of weight gain between 20 and 40 days of age, decreased oxidative stress at 60 days of age and did not influence the function of PMNLs in piglets.

Caracterização de genes de reparo de DNA em cana-de-açúcar

Sugarcane is an important culture for Brazil that holds almost half of all worldwide productivity. Plants face many challenges, because of biotic and abiotic stresses presents in the production field, which could prevent plants from reaching their genetic potential. As consequence, those stresses can generate Reactive Oxygen Species – ROS – that can cause damages on DNA. Another consequence of stress is the early-flowering process, which contributes for a reduction on yield. In this context, the aim of this work is to characterize ScMUTM1 and ScMUTM2, two DNA glycosylases belonging to base excision repair pathway; and identify genes potentially related to stress and DNA repair in two sugarcane cultivars with contrasting flowering phenotypes. The characterization of the DNA glycosylases included the construction of vector to over express the recombinant proteins ScMUTM1 and ScMUTM2; they will be used in a near future to purification of these proteins and use in enzymatic assays. It was also made a phylogenetic reconstruction of this gene in plants and analysis of its promoter. With the phylogenetic analysis, it is possible to observe the presence of these genes grouped inside a branch with monocots and another one with dicots. This suggests that the duplication of this gene probably occurred after the separation of these two groups. The analysis of the promotor of MUTM shows of the presence of stress-related regulatory motifs at ScMUTM2 promoter, when compared with ScMUTM1. This may suggests that ScMUTM1 might be suffering sub functionalization process. After the analysis of microarrays data, it is observed an up-regulation from some stress-related genes in one of the conditions analyzed, related to early flowering process.

Caracterização de genes de reparo de DNA em cana-de-açúcar

Sugarcane is an important culture for Brazil that holds almost half of all worldwide productivity. Plants face many challenges, because of biotic and abiotic stresses presents in the production field, which could prevent plants from reaching their genetic potential. As consequence, those stresses can generate Reactive Oxygen Species – ROS – that can cause damages on DNA. Another consequence of stress is the early-flowering process, which contributes for a reduction on yield. In this context, the aim of this work is to characterize ScMUTM1 and ScMUTM2, two DNA glycosylases belonging to base excision repair pathway; and identify genes potentially related to stress and DNA repair in two sugarcane cultivars with contrasting flowering phenotypes. The characterization of the DNA glycosylases included the construction of vector to over express the recombinant proteins ScMUTM1 and ScMUTM2; they will be used in a near future to purification of these proteins and use in enzymatic assays. It was also made a phylogenetic reconstruction of this gene in plants and analysis of its promoter. With the phylogenetic analysis, it is possible to observe the presence of these genes grouped inside a branch with monocots and another one with dicots. This suggests that the duplication of this gene probably occurred after the separation of these two groups. The analysis of the promotor of MUTM shows of the presence of stress-related regulatory motifs at ScMUTM2 promoter, when compared with ScMUTM1. This may suggests that ScMUTM1 might be suffering sub functionalization process. After the analysis of microarrays data, it is observed an up-regulation from some stress-related genes in one of the conditions analyzed, related to early flowering process.

Efeitos da terapia fotodinâmica com aluminio – cloro ftalocianina sobre a peroxidação lipídica e produtos antioxidantes em tecidos inflamados animais

Photodynamic therapy (PDT) consists of a non-toxic photosensitizing agent (FS) administration followed by a laser source resulting in a sequence of photochemical and photobiological processes that generate reactive oxygen species (ROS) that damaging cells. The present work evaluated the effects of PDT nanoemulsion-aluminum chloride phthalocyanine (AlClFc) mediated on malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD) and glutathione peroxidase (GPx) levels, which represent indicators involved in oxidative stress and antioxidant defenses. For this purpose, this study used 120 female rats of the Rattus norvegicus species, Wistar race, divided into 5 groups: Healthy (H), with periodontal disease (PD), with periodontal disease and treatment with FS (F), with periodontal disease and treatment with the laser (L); and periodontal disease and treatment with PDT (FL). An experimental model for represent periodontal disease (PD) was induced by ligature (split-mouth). Seven days later the induction of PD, the treatments were instituted according to the groups. In the group treated with PDT was applied 40μl FS (5μM) followed by laser irradiation diode InGaAlP (660nm, 100J / cm2). The rats were sacrificed on the 7th and 28th day after treatment and tissue specimens were removed and subjected to histological, immunohistochemical methods and enzymatic colorimetric measurements with detection by UV / VIS spectroscopy. Inflammatory changes, connective tissue disorganization and alveolar bone loss were displaying in groups with PD induced. The enzyme dosages showed that MDA levels were higher in PD induced groups, with no statistically significant differences (p> 0.05). High levels of GSH were found in groups L (p = 0.028) and FL (p = 0.028) compared with PD group, with statistically significant differences. Immunohistochemistry for SOD showed higher immunostaining in L and FL groups, compared to the PD group without statistically significant differences (p> 0.05). GPx showed lower immunoreactivity in the DP group when compared to the other groups and statistically significant differences were observed between the DPxL groups (p <0.05). TFD administered in this experiment did not induce elevation of MDA levels significantly increased the GSH levels and showed intense immunostaining pada SOD and GPx, showing that this therapy does not accentuated lipid peroxidation, however, it was able to induce effects on the antioxidant defenses processes. The LBI therapy appeared to show photomodulatory promoting effects reduction of the MDA levels, increasing GSH levels and with intense immunostaining for SOD and GPx, demonstrating that laser therapy induced antioxidant effects.

Efeito da exposição ao herbicida glifosato sobre parâmetros bioquímicos, moleculares e espermáticos do peixe Danio rerio

Agroquímicos são amplamente utilizados na atividade agrícola com o objetivo de aumentar a produção e melhorar a qualidade dos alimentos, no entanto podem vir a gerar danos ao meio ambiente e a organismos não-alvo. Dentre esses pesticidas encontra-se o herbicida glifosato, o qual vem sendo mais utilizado mundialmente. Seu mecanismo de ação se dá através da inibição da enzima 5-enolpiruvilshikimato-3- fosfatosintase, intermediária da síntese de aminoácidos aromáticos essenciais em plantas. Pouco se sabe sobre os efeitos da substância glifosato em animais, pois os estudos realizados visam principalmente os efeitos da formulação comercial, a qual contém surfactantes e outras substâncias inertes. Tendo isso em vista, esse estudo avaliou o efeito do glifosato no teleósteo Danio rerio considerando parâmetros de estresse oxidativo, atividade e expressão da acetilcolinesterase e parâmetros reprodutivos. Foram feitas exposições a 5 mg/L e 10 mg/L de glifosato, mais um grupo controle por 24 e 96 horas, somente com peixes machos. Para análise bioquímica foram retirados cérebro, brânquias e músculo; para análise molecular, cérebro e músculo; e para análise na qualidade espermática dos peixes, os testículos. Quanto às análises bioquímicas houve um aumento na capacidade antioxidante contra radicais peroxil nas brânquias na concentração de 5 mg/L após 24 horas de exposição; uma redução na peroxidação lipídica no cérebro na maior concentração (10 mg/L) após 24h e um aumento da mesma em músculo, também em 10 mg/L, após 96 horas. Não foi observada alteração na geração de espécies reativas de oxigênio decorrente da exposição ao glifosato, assim como na atividade da enzima acetilcolinesterase; já na expressão gênica desta enzima houve uma diminuição no cérebro após 24 horas de exposição e um aumento no cérebro e no músculo após 96 horas. Quanto à qualidade espermática dos peixes, houve uma redução na motilidade e período de motilidade dos espermatozóides nas concentrações de 5 mg/L e 10 mg/L em ambos tempos de exposição; na concentração de 10 mg/L ainda houve uma redução da funcionalidade mitocondrial, integridade de membrana do espermatozóide e integridade de DNA após 24 e 96 horas. Sendo assim, o glifosato se mostrou capaz de alterar o balanço oxidativo dos tecidos do peixe Danio rerio bem como alterar significativamente a expressão gênica da enzima acetilcolinesterase. Além disso, nossos resultados demonstram que o glifosato pode interferir na reprodução deste animal, através da redução de sua qualidade espermática.

Parâmetros do metabolismo oxidativo na anêmona-do-mar Bunodosoma cangicum: variação sazonal e efeitos da exposição ao ar

Bunodosoma cangicum é uma anêmona-do-mar que habita a faixa intermarés nas regiões sul e sudeste do Brasil. Assim como outros animais característicos destes locais, esta espécie de anêmona enfrenta diariamente as mudanças nos parâmetros ambientais decorrentes do ciclo de marés, os quais podem variar conforme a estação do ano. Estas mudanças podem alterar o metabolismo oxidativo dos animais destes habtats, que pode também ser influenciado pelas diferentes estações ao longo do ano. Portanto, a influência de dois períodos distintos durante o ano além da exposição ao ar sobre parâmetros oxidativos (Capacidade antioxidante total contra radicais peroxil - ACAP, atividade da glutamato cisteína ligase - GCL, conteúdo de glutationa reduzida - GSH e nível de perxidação lipídica - LPO) foi avaliada em anêmonas-do-mar coletadas em situação de submersão ou de emersão em um perío do frio e um quente (final de inverno/começo de primavera e início de outono). A resposta destes parâmetros, bem como do conteúdo de espécies reativas de oxigênio (ERO) e de adenosina trifosfato (ATP), também foi avaliada em animais submetidos diariamente à exposição ao ar (3 h) em laboratório por 30 dias. Com relação aos parâmetros oxidativos considerando apenas os diferentes períodos do ano, uma maior atividade da GCL foi observada durante o período mais frio, assim como um maior nível de LPO neste mesmo período. Com relação à exposição ao ar, no que diz respeito às defesas antioxidantes, em animais coletados em emersão foi observado uma maior atividade da GCL durante o período quente, além de uma maior ACAP e um menor conteúdo de GSH em anêmonas-do-mar coletadas, tanto no período frio como quente. Com relação aos danos oxidativos, um maior nível de LPO foi encontrado em anêmonas-do-mar coletadas durante emersão no período mais (outono). De forma geral, não foi observado um padrão de variação dos parâmetros oxidativos em função da hora do dia, evidenciando-se apenas uma diminuição na ACAP e um aumento da GSH, em torno das 13h, em animais coletados durante a estação mais quente. As 7 anêmonas-do-mar expostas ao ar sob condições controladas de laboratório mostraram variações transitórias da ACAP (aumento) e do conteúdo de GSH (redução) após a reoxigenação. Estes resultados indicam que alguns parâmetros oxidativos de B. cangicum apresentam variação sazonal enquanto outros são afetados pela exposição ao ar. No entanto, o padrão de resposta destes parâmetros é diferente em campo e em laboratório, sugerindo que os parâmetros controlados em laboratório, tais como temperatura, fotoperíodo e iluminação, modificam a resposta do metabolismo oxidativo de B. cangicum à exposição ao ar em campo.

Association between common lipid metabolism genes polymorphisms and sporadic colorectal adenocarcinoma risk

Lipids can modulate the risk of developing sporadic colorectal adenocarcinoma (SCA), since alterations into lipid metabolism and transport pathways influence directly cholesterol and lipids absorption by colonic cells and indirectly reactive oxygen species (ROS) synthesis in rectum cells due to lipid accumulation. Lipid metabolism is regulated by several proteins APOA1, APOB, APOC3, APOE, CETP, NPY, PON1 and PPARG that could influence both metabolism and transport processes. Is been reported that several common single-nucleotide polymorphisms (SNPs) in these genes could influence their function and/or expression, changing lipid metabolism balance. Thus, genetic changes in those genes can influence SCA development, once the majority of them were never studied in this disease. Furthermore, there are contradictory results between some studied polymorphisms and SCA risk. Thus, the aim of this study was to explore and describe lipid metabolism-associated genes common polymorphisms (APOA1 -75 G>A; APOB R3500Q; APOC3 C3175G, APOC3 T3206G; APOE Cys112/158Arg; CETP G279A, CETP R451Q; NPY Leu7Pro; PON1 Q192R; PPARG Pro12Ala) status among SCA, and their relationship with SCA risk. Genotyping of common lipid metabolism genes polymorphisms (APOA1 75 G>A; APOB R3500Q; APOC3 C3175G, APOC3 T3206G; APOE Cys112/158Arg; CETP G279A, CETP R451Q; NPY Leu7Pro; PON1 Q192R; PPARG Pro12Ala) were done by PCR-SSP techniques, from formalin-fixed and paraffin-embedded biopsies of 100 healthy individuals and 68 SCA subjects. Mutant genotypes of APOA1 -75AA (32% vs 12%; p=0.001; OR=3.51; 95% CI 1.59-7.72); APOB 3500AA (7% vs 0%; p=0.01); APOC3 3175GG (19% vs 2%; p=0.0002; OR=11.58; 95% CI 2.52-53.22), APOC3 3206GG (19% vs 0%; p<0.0001); CETP 279AA (12% vs 1%; p=0.003; OR=13.20; 95% CI 1.61-108.17), CETP 451AA (16% vs 0%; p<0.0001); NPY 7CC (15% vs 0%; p<0.0001); PPARG 12GG (10% vs 0%; p=0.001); and heterozygote genotype PON1 192AG (56% vs 22%; p<0.0001; OR=4.49; 95% CI 2.298.80) were found associated with SCA prevalence. While, APOE E4/E4 (0% vs 8%; p=0.02) mutant haplotype seemed to have a protective effect on SCA. Moreover, it also been founded differences between APOB 3500GA, APOC3 3206TG, CETP 279AA genotypes and PPARG 12Ala allele prevalence and tissue localization (colon vs rectum). These findings suggest a positive association between most of common lipid metabolism genes polymorphisms studied and SCA prevalence. Dysregulation of APOA1, APOB, APOC3, CETP, NPY, PON1 and PPARG genes could be associated with lower cholesterol plasma levels and increase ROS among colon and rectum mucosa. Furthermore, these results also support the hypothesis that CRC is related with intestinal lipid absorption decrease and secondary bile acids production increase. Moreover, the polymorphisms studied may play an important role as biomarkers to SCA susceptibility.

Association between common lipid metabolism genes polymorphisms and sporadic colorectal adenocarcinoma risk

Lipids can modulate the risk of developing sporadic colorectal adenocarcinoma (SCA), since alterations into lipid metabolism and transport pathways influence directly cholesterol and lipids absorption by colonic cells and indirectly reactive oxygen species (ROS) synthesis in rectum cells due to lipid accumulation. Lipid metabolism is regulated by several proteins APOA1, APOB, APOC3, APOE, CETP, NPY, PON1 and PPARG that could influence both metabolism and transport processes. Is been reported that several common single-nucleotide polymorphisms (SNPs) in these genes could influence their function and/or expression, changing lipid metabolism balance. Thus, genetic changes in those genes can influence SCA development, once the majority of them were never studied in this disease. Furthermore, there are contradictory results between some studied polymorphisms and SCA risk. Thus, the aim of this study was to explore and describe lipid metabolism-associated genes common polymorphisms (APOA1 -75 G>A; APOB R3500Q; APOC3 C3175G, APOC3 T3206G; APOE Cys112/158Arg; CETP G279A, CETP R451Q; NPY Leu7Pro; PON1 Q192R; PPARG Pro12Ala) status among SCA, and their relationship with SCA risk. Genotyping of common lipid metabolism genes polymorphisms (APOA1 75 G>A; APOB R3500Q; APOC3 C3175G, APOC3 T3206G; APOE Cys112/158Arg; CETP G279A, CETP R451Q; NPY Leu7Pro; PON1 Q192R; PPARG Pro12Ala) were done by PCR-SSP techniques, from formalin-fixed and paraffin-embedded biopsies of 100 healthy individuals and 68 SCA subjects. Mutant genotypes of APOA1 -75AA (32% vs 12%; p=0.001; OR=3.51; 95% CI 1.59-7.72); APOB 3500AA (7% vs 0%; p=0.01); APOC3 3175GG (19% vs 2%; p=0.0002; OR=11.58; 95% CI 2.52-53.22), APOC3 3206GG (19% vs 0%; p<0.0001); CETP 279AA (12% vs 1%; p=0.003; OR=13.20; 95% CI 1.61-108.17), CETP 451AA (16% vs 0%; p<0.0001); NPY 7CC (15% vs 0%; p<0.0001); PPARG 12GG (10% vs 0%; p=0.001); and heterozygote genotype PON1 192AG (56% vs 22%; p<0.0001; OR=4.49; 95% CI 2.298.80) were found associated with SCA prevalence. While, APOE E4/E4 (0% vs 8%; p=0.02) mutant haplotype seemed to have a protective effect on SCA. Moreover, it also been founded differences between APOB 3500GA, APOC3 3206TG, CETP 279AA genotypes and PPARG 12Ala allele prevalence and tissue localization (colon vs rectum). These findings suggest a positive association between most of common lipid metabolism genes polymorphisms studied and SCA prevalence. Dysregulation of APOA1, APOB, APOC3, CETP, NPY, PON1 and PPARG genes could be associated with lower cholesterol plasma levels and increase ROS among colon and rectum mucosa. Furthermore, these results also support the hypothesis that CRC is related with intestinal lipid absorption decrease and secondary bile acids production increase. Moreover, the polymorphisms studied may play an important role as biomarkers to SCA susceptibility.

Modulação da expressão da proteína XPA em resposta ao tratamento com azul de metileno fotossensibilizado

Reactive oxygen species (ROS) are produced by aerobic metabolism and react with biomolecules, such as lipids, proteins and DNA. In high concentration, they lead to oxidative stress. Among ROS, singlet oxygen (1O2) is one of the main ROS involved in oxidative stress and is one of the most reactive forms of molecular oxygen. The exposure of some dyes, such as methylene blue (MB) to light (MB+VL), is able to generate 1O2 and it is the principle involved in photodynamic therapy (PDT). 1O2 e other ROS have caused toxic and carcinogenic effects and have been associated with ageing, neurodegenerative diseases and cancer. Oxidative DNA damage is mainly repaired by base excision repair (BER) pathway. However, recent studies have observed the involvement of nucleotide excision repair (NER) factors in the repair of this type of injury. One of these factors is the Xeroderma Pigmentosum Complementation Group A (XPA) protein, which acts with other proteins in DNA damage recognition and in the recruitment of other repair factors. Moreover, oxidative agents such as 1O2 can induce gene expression. In this context, this study aimed at evaluating the response of XPA-deficient cells after treatment with photosensitized MB. For this purpose, we analyzed the cell viability and occurrence of oxidative DNA damage in cells lines proficient and deficient in XPA after treatment with MB+VL, and evaluated the expression of this enzyme in proficient and complemented cells. Our results indicate an increased resistance to treatment of complemented cells and a higher level of oxidative damage in the deficient cell lines. Furthermore, the treatment was able to modulate the XPA expression up to 24 hours later. These results indicate a direct evidence for the involvement of NER enzymes in the repair of oxidative damage. Besides, a better understanding of the effects of PDT on the induction of gene expression could be provided

Células de anêmonas Bunodosoma cangicum expostas ao cobre: citotoxidade, defesa e dano de DNA

Anêmonas-do-mar são pólipos solitários, bentônicos, de pouca mobilidade, que habitam regiões entre-marés. Devido a estas características, são organismos que podem ser atingidos diretamente pela poluição aquática, no entanto, são pouco utilizados como modelo ecotoxicológico. O cobre é um metal essencial, que em altas concentrações pode ser tóxico, sendo bastante comum em ecossistemas marinhos. Um dos mecanismos de toxicidade do cobre envolve a produção de espécies reativas de oxigênio (ERO), podendo levar as células ao estresse oxidativo, que tem como característica danos celulares, inclusive no DNA. Muitos organismos possuem um mecanismo que bombeia os xenobióticos para fora da célula – multixenobiotic resistance (MXR) – que visa prevenir as células dos danos tóxicos causados pelo contaminante. Com isso, o presente trabalho estudou a capacidade de defesa e dano ao DNA à toxicidade causada pelo cobre em células de anêmonas Bunodosoma cangicum. Para isto, células de anêmonas, mantidas em cultura primária através de explante do disco podal, foram expostas ao cobre a duas concentrações (7,8 µg.L-1 Cu e 15,6 µg.L-1 Cu), além do grupo controle, por 6 e 24 h. Antes e após as exposições as células tiveram sua viabilidade avaliada através do método de exclusão por azul de tripan (0,08%) para analisar a citotoxicidade. Parâmetros como a indução do mecanismo MXR através do método de acúmulo de rodamina-B, espécies reativas de oxigênio e ensaio cometa, também foram avaliados. Os resultados obtidos mostram que o cobre é citotóxico, sendo constatada uma queda na viabilidade e no número de células, principalmente após 24 h de exposição, sendo que na concentração de cobre de 15,6 µg.L-1 , foi possível observar uma diminuição de 40% na viabilidade e uma redução em 36% no número de células (p < 0,05, n = 6). Em relação ao fenótipo MXR, foi observada uma ativação do mecanismo apenas naquelas células expostas ao cobre 7,8 µg.L-1 (53%) no tempo de 24 h (p < 0,05, n = 5). Na análise da geração de ERO foi observado um aumento de 11,5% naquelas células expostas por 6 h na concentração mais alta de cobre 15,6 µg.L-1 . Nas células que foram expostas por 24 h, o aumento de espécies reativas pode ser percebido já na concentração de 7,8 µg.L-1 , elevando-se para cerca de 20% quando exposto a 15,6 µg.L-1 (p < 0,05, n = 4-5). Quanto ao dano de DNA, foram vistas quebras na molécula desde 7,8 µg.L-1 Cu em 6 h, com danos ainda mais salientes naquelas células expostas por 24 h, na concentração de 7,8 µg.L -1 Cu (p < 0,05, n = 3-4), e para 15,6 µg.L-1 Cu a viabilidade celular (número de células) não permitiu a análise. Com base nestes dados, pode-se dizer que o cobre, mesmo em baixas concentrações causa estresse em células de B. cangicum, sendo citotóxico. Este metal causa estresse oxidativo com dano à molécula de DNA mesmo com a ativação do mecanismo de defesa.

Coordenação entre as respostas da catalase e da ascorbato peroxidade em folhas de feijão caupi submetidas a diferentes fontes de peróxido de hidrogênio

The plants are often exposed to variations in environmental conditions that may trigger metabolic disturbances leading to a consequent loss in productivity of crops. These stressful conditions usually induce an accumulation of reactive oxygen species (ROS) in the cell, a condition known how oxidative stress. Among these species, hydrogen peroxide (H2O2) is an important molecule involved in numerous signaling mechanisms. The present study aimed to understand the relationship between the different enzymatic mechanisms of elimination of H2O2 by catalase (CAT) and ascorbate peroxidase (APX) in leaf tissues of seedlings of the species Vigna unguiculata L. Walp, under conditions of oxidative stress induced by application of CAT inhibitor, 3-amino-1,2,4-triazole (3-AT), and H2O2 itself on the roots. Three experiments were conducted. The first experiment was performed applying the compound 3-AT (5 mM) during the time (hours). In the second experiment, seedlings were exposed to different concentrations of H2O2 (2.5, 5.0, 7.5, 10 mM) for 48 h. The third strategy included the pre-treatment with H2O2 (2.5 mM) for 24 h, followed by subsequent treatment with the inhibitor 3-AT and recovery control condition. Treatment with 3-AT causes a strong inhibition of CAT activity in leaf tissues accompanied by an increase of activity of APX. However a decrease in oxidative damage to lipids is not observed as indicated by TBARS. It was observed that activity of APX is directly linked to the content of peroxide. Inductions in the activities of CAT and APX were observed mainly in the seedlings treated with 2.5 mM H2O2. This can be associated with a decrease in oxidative damage to lipids. In contrast, one same tendency was not observed in treatments with higher concentrations of this ROS. These results suggest that the concentration of 2.5 mM H2O2 can induce responses antioxidants later in seedling cowpea. This concentration when applied as pre-treatment for 24 h promoted an induction systems removers CAT and APX, both in activity and in terms of gene expression. However this increment was not observed in the recovered plants and the plants subsequently subjected to 3-AT. Additionally, the pretreatment was not sufficient to attenuate the inhibition of CAT activity and oxidative damage to lipids caused by the subsequent application of this inhibitor. The results showed that the application of 3-AT and H2O2 in the root systems of seedlings of cowpea promote changes in the parameters analyzed in leaf tissues that indicate a direct response to the presence of these factors or systemic signaling mecanisms. H2O2 appears to activate the responses of two antioxidant systems in this study thar does not promote greater protection in case of additional treatment with 3-AT. This demonstrates the importance of the CAT system. In this work, complete results indicate that there is a difference between the signaling and the effects caused by exposure to H2O2 and by treatment with 3-AT

Análise da expressão de APE1 após estresse oxidativo induzido em células proficientes e deficientes na via de reparo por excisão de nucleotídeos.

Riboflavin is a vitamin very important in aerobic organisms, as a precursor of many coenzymes involved in the electron transporter chain. However, after photosensitization of riboflavin with UV or visible light, it generates reactive oxygen species (ROS), which can oxidize the DNA. The repair of oxidative lesions on DNA occurs through the base excision repair pathway (BER), where APE1 endonuclease plays a central role. On the other hand, the nucleotide excision repair pathway (NER) repairs helix-distorting lesions. Recently, it was described the participation of NERproteins in the repair of oxidative damage and in stimulation of repair function fromAPE1. The aim of this research was to evaluate the cytotoxic effects of photosensitized riboflavin (RF*) in cells proficient and deficient in NER, correlating with APE1 expression. For this propose, the cells were treated with RF* and it was performed the cell viability assay, extraction of whole proteins, cells fractionation, immunoblotting, indirect immunofluorescence and analysis of polymorphisms of BER gens. The results evidenced that cells deficient in XPA and CSB proteins were more sensitive to RF*. However, XPC-deficient cells presented similar resistance to MRC5- SV cells, which is proficient in NER. These results indicate that XPA and CSB proteins have an important role on repair of oxidative lesions induced by RF*. Additionally, it was evidenced that single nucleotide polymorphisms (SNPs) in BER enzymes may influence in sensitivity of NER-deficient cell lines. Concerning the APE1 expression, the results showed that expression of this protein after treatment with RF* only changed in XPC-deficient cells. Though, it was observed that APE1 is recruited and is bound to chromatin in MRC5-SV and XPA cells after treatment with RF*. The results also showed the induction of DNA damage after treatment with RF*, through the analysis of-H2AX, since the treatment promoted an increase of endogenous levels of this phosphorylated protein, which acts signaling double strand-break on DNA. On the other hand, in XPC-deficient cells, regardless of resistance of RF*, the endogenous levels of APE1 are extremely reduced when compared with other cell lines and APE1 is not bound to chromatin after treatment with RF*. These results conclude that RF* was able to induce cell death in NERdeficient cells, where XPA and CSB cells were more sensitive when compared with MRC5-SV and XPC-deficient cells. This last result is potentially very interesting, since XPC-deficient cell line presents low levels of APE1. Additionally, the results evidenced that APE1 protein can be involved in the repair of oxidative damage induced by RF*, because APE1 is recruited and bound strongly to chromatin after treatment.

Medida da capacidade antioxidante de fluidos biologicos por voltametria de pulso diferencial associada ao uso de surfactante como resposta as adaptações geradas pelo exercicio

Universidade Estadual de Campinas . Faculdade de Educação Física

O efeito da restrição calórica na longevidade

A restrição calórica (RC) é uma das formas de intervenção nutricional mais amplamente discutida para se estender o tempo de vida em uma variedade de espécies, inclusive seres humanos. A RC parece reduzir a incidência de doenças relacionadas à idade. O mecanismo clássico que poderia explicar o efeito do consumo calórico no envelhecimento está relacionado à redução da gordura corporal e à sinalização da insulina, somada às espécies reativas de oxigênio produzidas durante a respiração que causam danos oxidativos ao DNA e ao RNA das células, promovendo o processo de envelhecimento e o aumento do risco de doenças. No entanto, o efeito da RC na longevidade em humanos ainda não está bem estabelecido e mais estudos são necessários para que os mecanismos celulares e moleculares responsáveis pelos efeitos terapêuticos da restrição calórica sejam elucidados. Além disso, é necessário diferenciar os efeitos benéficos da restrição calórica daqueles relacionados a hábitos alimentares saudáveis.