As células trofoblásticas provenientes de cones ectoplacentários na fase de pós-implantação expressam as subunidades do complexo NAD(P)H-oxidase de membrana p22-phox, gp91-phox, p47-phox, p40-phox, p67-phox e Rac1 previamente descritas em fagócitos e envolvidas com a atividade fagocitária destas células. Ocorre modulação da expressão das subunidades do complexo enzimático NAD(P)H-oxidase sob o estímulo com PMA, agente capaz de fosforilar a subunidade p47-phox e ativar esta enzima em fagócitos profissionais. Nossos ensaios experimentais indicam que as células trofoblásticas são capazes de gerar espécies reativas de oxigênio dependente da atividade do complexo NAD(P)H-oxidase de membrana de baixa intensidade quando não estimuladas e de forma muito mais intensa quando estimuladas pelo PMA. As características de expressão e atividade do complexo enzimático NAD(P)H-oxidase de membrana encontradas neste estudo sugerem que esta enzima é semelhante a encontrada em outros fagócitos e, desta forma possa talvez estar também envolvida com processos de defesa da interface materno-fetal. / In macrophages and neutrophils, the activation of the phagocytosis is defined by the acquisition of competence for microbicide, tumoricide and cytolytic functions resultant of generation and release of oxygen reactive species, besides the phagocytic process per se. Like these phagocytes, trophoblast cells in many species are also phagocytic. This cellular population involves completely the embryo and exhibits different and specific characteristics along the gestation. In rodents and primates, these cells are strategically positioned between maternal and fetal circulation and exhibit invasive and phagocytic activity, respectively responsible for anchorage of the embryo into the endometrium and uptake of an adequate nutritional supply for the embryo development. In rodents, these activities present a maximum degree during the implantation period, gradually declining, as the placenta develops. In the presence of strange particles at the maternal-placental interface, however, this process can be reactivated and, in this case, may be related to defense\'s mechanisms. Previous studies performed in our laboratory showed the potential of the trophoblast in producing and releasing reactive species of oxygen/nitrogen, in a very similar manner to that observed in macrophages and neutrophils. The production of such molecules is associated to different enzymes, but the localization of hydrogen peroxide on trophoblast cell surface has suggested a NAD(P)H-oxidase activity. NAD(P)H-oxidase is formed by the cytochrome b558 associated to the cellular membrane (subunits p22-phox + gp91-phox), the cytosolic subunits p47-phox, p67-phox and p40phox and, the GTPases Rac1 and Rac2 in an electron generator system that uses NADH or NADPH as substratum. Once activated, the enzymatic complex is responsible for the electron inflow to the molecular oxygen, yields superoxide anion. Thus, based on the literature and results previously obtained by our group, this study analyzed the protein and gene expression of the NAD(P)H-oxidase complex subunits respectively by immunolocalization and Westernblotting and rt-PCR, in the mouse trophoblast stimulated with PMA. Rt-PCR semi-quantitative analyses showed increase expression of the subunits p22-phox, gp91-phox, p47-phox, p67-phox, p40phox and Rac1 in PMA-treated in comparison with non treated ectoplacental cones. The expression of the subunits gp91-phox, p47-phox and p67-phox were confirmed by Western blotting and, like gene expression also increased in the presence of PMA. These subunits were mostly located in the trophoblast giant cell population, associated to the phagocytic process at the maternal-placental interface. Increased expression of such subunits may be related to an increase in the NAD(P)H-oxidase activity. To analyze this possibility and to determine the role played by NAD(P)H-oxidase activity in the reactive oxygen species produced by trophoblast cells, cellular assays were performed using the oxyethidium fluorescence, a product of dihydroethidium oxidation by superoxide anion. Thus, under PMA stimulus and antimycin A that blocks the mitochondrial NAD(P)H-oxidase activity and, apocynin and allopurinol, respectively blocking the membrane NAD(P)H-oxidase and xhantine oxidase and, still, using specific superoxide and hydrogen peroxide scavengers (superoxide dismutase enzyme and catalase) we showed the generation of reactive species of oxygen-NAD(P)H-oxidase dependent by trophoblast cells, mostly when stimulated. These results come to add important information about the potential of the trophoblast in producing reactive species at the maternal-fetal interface and, open a new investigation interest on the NADPH-oxidase regulatory processes and its involvement in defense functions of the embryo in both healthy and pathological processes that can determine the failure of the gestation.

Economically advantageous alternatives to fully explore the antioxidant content of tropical fruit residues from juice processor industries have been currently studied in the State of Alagoas, Brazil. Thus, flour methanolic extracts of acerola (AC), passion fruit (PF) and pineapple (PA) residues were screened for their antioxidant capacity. For this, their total phenolic content, antioxidant capacity toward DPPH and toward the superoxide anion radical, together with analysis of their protective effect against membrane lipid peroxidation, caused by peroxyl radical, were determined in the flour methanolic extracts. In the last case, the phospholipid bilayer biomimetic model with the inclusion of a fluorescent probe was used. The statistical analysis employed the epi info version 6.04d (2001) program and Microsoft excel, using the test t, multivaried analysis and qui-square, considering p < 0.05 as significant. The results of total phenolic content of flour methanolic extracts of AC, PF and PA were 94.6 ± 7.4, 41.2 ± 4.2 and 9.1 ± 1.3 mg of gallic acid equivalents/ g of dry extract, respectively. AC showed the best total phenolic content and antioxidant capacity toward DPPH . PF showed the best antioxidant capacity to superoxide anion radical. Similar indexes of lipid peroxidation protection were observed with both AC and PF residues. Altogether, these data suggest that flour methanolic extracts of AC and PF residues may be useful as antioxidant supplements or food additives, notably the acerola (AC) extracts. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Alternativas economicamente vantajosas com exploração completa do conteúdo antioxidante de resíduos de frutas tropicais de fábricas de sucos foram estudadas no estado de Alagoas, Brasil. Assim, extratos metanólicos de farinhas de resíduos de acerola (FRAC), maracujá (FRMA) e abacaxi (FRAB) foram avaliados quanto a sua capacidade antioxidante. Para isso, foram analisados o conteúdo total de fenóis, a capacidade antioxidante frente aos radicais DPPH e ânion radical superóxido e o efeito de proteção à peroxidação da membrana lipídica, causada pelo radical peroxila, dos extratos metanólicos das farinhas. No ultimo caso, um modelo de membrana bilamelar fosfolipídica com uma sonda fluorescente foi usado. Para a análise estatística, foi utilizado o programa epi info versão 6.04d (2001) e o Microsoft Excel, usando teste t, análise multivariada e qui-quadrado, considerando um p < 0,05 como significativo. Os resultados para o conteúdo total de fenóis dos extratos metanólicos das farinhas de resíduos de acerola (FRAC), maracujá (FRMA) e abacaxi (FRAB) foram 94,6 ± 7,4, 41,2 ± 4,2 e 9,1 ± 1,3 mg de equivalentes de ácido gálico/ g de extrato seco, respectivamente. FRAC mostrou o melhor conteúdo de fenóis e capacidade antioxidante frente ao radical DPPH . FRMA mostrou a melhor capacidade antioxidante frente ao ânion radical superóxido. Resposta similar foi observada entre FRAC e FRMA quanto à proteção frente a peroxidação da membrana lipídica. De modo geral, estes dados sugerem que extratos metanólicos de farinhas de FRAC e FRMA podem ser úteis como suplementos antioxidantes ou aditivos alimentares, em especial, o extrato de acerola.