Atividades catalíticas de diferentes isoformas do citocromo P450 na pele e no fígado de toninhas, Pontoporia blainvillei (Gervais e D'Orbigny, 1844).

TCC(graduação) - Universidade Federal de Santa Catarina. Centro de Ciências Biológicas. Licenciatura em Ciências Biológicas.Os xenobióticos são compostos não sintetizados pelos organismos e que, portanto, não participam de processos fisiológicos. Dentre os xenobióticos mais estudados na ecotoxicologia podem-se citar os hidrocarbonetos policíclicos aromáticos, uma das classes de moléculas que compõem o petróleo e que estão classicamente relacionados com a indução de citocromos P450 (CYPs). Devido ao seu papel na biotransformação de xenobióticos, CYPs são importantes biomarcadores de exposição. Biomarcadores são importantes ferramentas utilizadas em programas de monitoramento ambiental que, ao indicarem o provável contato de indivíduos com xenobióticos, podem sinalizar a necessidade de conservação e manejo do ambiente natural e, assim, favorecer a preservação de diversas espécies, como a toninha. Pontoporia blainvillei, popularmente conhecida como toninha, é o cetáceo mais ameaçado do Sudoeste Atlântico. Por conta de seu pequeno tamanho, são frequentemente mortas em redes de pesca. Além disso, a degradação de seu ambiente natural em decorrência de dejetos de indústrias, esgoto sanitário e do tráfego de barcos também dificultam a conservação dessa espécie. O presente estudo visou avaliar a atividade e as propriedades cinéticas de enzimas citocromo P450 em P. blainvillei através da mensuração de atividades Alcóxi-resorufina Odeetilases (ARODs) (MROD, BROD e EROD) nos microssomas obtidos de amostras da pele e do fígado de indivíduos da espécie. Para cada atividade enzimática foram calculados os Kmapp e Vmáx, com base em uma curva cinética com concentrações crescentes de substrato. Ensaios com inibidores elipticina e cetoconazol foram efetuados para a investigação da prevalência de isoformas de CYPs em cada matriz tecidual. No fígado, foram detectadas atividades EROD, MROD e BROD. A atividade MROD apresentou Kmapp=0,6399 μM e Vmáx=3,592 pmol.min-1.mgprt-1; BROD apresentou Kmapp=0,6741 μM e Vmáx=0,2369 pmol.min-1.mgprt-1 e EROD apresentou Kmapp=0,2429 μM e Vmáx=1,602 pmol.min-1.mgprt-1 sendo, no fígado, todas atividades catalíticas melhores ajustadas ao modelo de inibição por substrato. Na pele, apenas atividades MROD e BROD foram detectadas, MROD com Kmapp=1,004 μM e Vmáx=75,64 fmol.min-1.mgprt-1, ajustando-se melhor ao modelo de inibição por substrato e BROD com Kmapp=0,6741 μM e Vmáx=0,2369 fmol.min-1.mgprt-1, ajustando-se melhor ao modelo de Michaelis-Menten. As concentrações ótimas para futuros ensaios no fígado foram: 1 mM de NADPH e entre 1 e 1,4 μM de 7-MR para a atividade MROD; 1 mM de NADPH e entre 2 e 2,5 μM de 7-BR para a BROD e 1 mM de NADPH e entre 0,8 e 1,57 μM de 7-ER para a EROD. Na pele as concentrações ótimas foram: 1 mM de NADPH e entre 0,15 a 0,25 μM de 7-MR para MROD e 0,25 mM de NADPH e entre 0,5 e 1 μM de 7-BR para a BROD. O inibidor de CYP1A1 (elipticina) inibiu completamente a atividade EROD e 52% da atividade BROD no fígado, sugerindo uma forte participação dessa isoforma na atividade EROD e BROD no fígado. A elipticina não inibiu atividade BROD na pele, já o cetoconazol, inibidor de CYP3A, inibiu 95,5% da atividade BROD no fígado e 79% na pele, indicando que isoformas da família 3A são relevantes para atividade BROD em ambos os tecidos de P. blainvillei.Xenobiotics are compounds that are not synthesized by organisms and, therefore, do not participate in physiological processes. Among the most studied xenobiotics in ecotoxicology, we can mention the PAHs, one of the classes of molecules that make up petroleum and that are classically related to the induction of cytochromes P450 (CYPs). Due to their role in the biotransformation of xenobiotics, CYPs are important biomarkers of exposure. Biomarkers are important tools used in environmental monitoring programs that, by indicating the probable contact of individuals with xenobiotics, can signal the need for conservation and management of the natural environment and thus favor the preservation of several species, such as the franciscana dolphin. Pontoporia blainvillei, popularly known as franciscana is the most threatened cetacean in the Southwest Atlantic. Because of their small size, they are often killed in fishing nets. In addition, the degradation of its natural environment because of waste from industries, sewage and boat traffic, also hinder the conservation of this species. The present study evaluated the activity and kinetic properties of cytochrome P450 enzymes in Pontoporia blainvillei by measuring ARODs activities (MROD, BROD and EROD) in liver and skin microsomes. For each enzyme activity, Kmapp and Vmax calculations were performed, based on a kinetic curvature with substrate crescents. Assays with ellipticin and ketoconazole inhibitors were performed to investigate the prevalence of CYPs isoforms in each tissue matrix. In liver, EROD, MROD and BROD activities were carried out. MROD activity showed Kmapp=0.6399 μM and Vmax=3.592 pmol.min-1.mgprt-1; BROD presented Kmapp=0.6741 μM and Vmax=0.2369 pmol.min-1.mgprt-1 and EROD presented Kmapp=0.2429 μM and Vmax=1.602 pmol.min-1.mgprt-1 being, in liver, all catalytic activities better adjusted to substrate inhibition model. In skin, only MROD and BROD activities were detected, MROD with Kmapp=1.004 μM and Vmax=75.64 fmol.min-1.mgprt-1, better fitting substrate inhibition model and BROD with Kmapp=0.6741 μM and Vmax=0.2369 fmol.min-1.mgprt-1, better fitting the Michaelis-Menten model. Optimal concentrations for future liver assays were: For MROD activity, 1 mM NADPH and 1 to 1.4 μM of 7-MR; BROD, 1 mM NADPH and between 2 to 2.5 μM of 7-BR and EROD, 1 mM of NADPH and between 0.8 to 1.57 μM of 7-ER. In skin, optimal concentrations were: for MROD 1 mM of NADPH and between 0.15 to 0.25 μM of 7-MR and for BROD 0.25 mM of NADPH and 0.5 to 1 μM of 7-BR. CYP1A1 inhibitor (ellipticin) completely inhibited EROD activity and inhibited 52% of BROD activity in the liver, suggesting a strong participation of this isoform in EROD and BROD activity in this organ. Ellipticin did not inhibit BROD activity in skin, whereas ketoconazole, a CYP3A inhibitor, inhibited 95.5% of BROD activity in liver and 79% in skin, indicating that family 3A isoforms are relevant for BROD activity in both tissues of Pontoporia blainvillei

NOx-, IL-1β-, TNF-α-, and IL-6-Inhibiting Effects and Trypanocidal Activity of Banana (Musa acuminata) Bracts and Flowers: UPLC-HRESI-MS Detection of Phenylpropanoid Sucrose Esters

Banana inflorescences are a byproduct of banana cultivation consumed in various regions of Brazil as a non-conventional food. This byproduct represents an alternative food supply that can contribute to the resolution of nutritional problems and hunger. This product is also used in Asia as a traditional remedy for the treatment of various illnesses such as bronchitis and dysentery. However, there is a lack of chemical and pharmacological data to support its consumption as a functional food. Therefore, this work aimed to study the anti-inflammatory action of Musa acuminata blossom by quantifying the cytokine levels (NOx, IL-1β, TNF-α, and IL-6) in peritoneal neutrophils, and to study its antiparasitic activities using the intracellular forms of T. cruzi, L. amazonensis, and L. infantum. This work also aimed to establish the chemical profile of the inflorescence using UPLC-ESI-MS analysis. Flowers and the crude bract extracts were partitioned in dichloromethane and n-butanol to afford four fractions (FDCM, FNBU, BDCM, and BNBU). FDCM showed moderate trypanocidal activity and promising anti-inflammatory properties by inhibiting IL-1β, TNF-α, and IL-6. BDCM significantly inhibited the secretion of TNF-α, while BNBU was active against IL-6 and NOx. LCMS data of these fractions revealed an unprecedented presence of arylpropanoid sucroses alongside flavonoids, triterpenes, benzofurans, stilbenes, and iridoids. The obtained results revealed that banana inflorescences could be used as an anti-inflammatory food ingredient to control inflammatory diseases