Avaliação da intervenção nutricional com vitaminas E e C, e o mineral zinco no estresse oxidativo de pacientes com hepatite C em tratamento com interferon associado à ribavirina

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências da Saúde. Programa de Pós-Graduação em Farmácia.As hepatites virais apresentam distribuição universal e magnitude variável de acordo com as regiões dos distintos países. A WHO estima que em torno de 3% da população mundial esteja infectada pelo vírus da hepatite C (VHC). Os mecanismos pelos quais o vírus da hepatite C causa dano celular ainda não são bem compreendidos, incluindo dano hepático imunológico, dano diretamente citotóxico mediado por diferentes produtos virais, bem como estresse oxidativo, têm sido sugeridos na patogênese da hepatite C crônica. O objetivo deste trabalho foi avaliar o status antioxidante no sangue de pacientes com hepatite C sem tratamento (Grupo II) e em tratamento com interferon peguilado associado à ribavirina (Grupo III) comparados a indivíduos saudáveis (Grupo I), antes e após suplementação antioxidante durante 6 meses (vitaminas E 800 mg/dia e C 500 mg/dia, e o mineral Zn 40 mg/dia). Foram examinadas as atividades das enzimas antioxidantes catalase (CAT), superóxido dismutase (SOD), glutationa peroxidase (GPx), glutationa redutase (GR) e glutationa S-transferase (GST), e as defesas antioxidantes não-enzimáticas, como os conteúdos de glutationa reduzida (GSH), vitamina E e o mineral Zn. Foram dosados também os marcadores de dano, como a lipoperoxidação (níveis de TBARS), proteína carbonilada (PC), atividades da gama-glutamiltransferase (GGT), da alanina aminotransferase (ALT), da aspartato aminotransferase (AST) e da fosfatase alcalina (ALP). Foram avaliadas ainda a atividade da mieloperoxidase (MPO) e a concentração de ferro (Fe) eritrocitário. Os resultados das análises dos biomarcadores enzimáticos de estresse oxidativo mostraram muitas alterações entre os três grupos avaliados. Nas determinações iniciais, observou-se aumento da atividade de algumas enzimas (CAT, SOD, MPO, ALT) e depleção da concentração de GSH e vitamina E em pacientes com hepatite C sem tratamento (Grupo II). Foram observados aumentos na atividade enzimática da CAT, GST e GR, na concentração de PC, bem como depleção dos conteúdos de GSH em pacientes com hepatite C em tratamento com interferon peguilado associado à ribavirina (Grupo III), tanto quando comparados ao controle negativo (banco de sangue-Grupo I), como quando comparados ao grupo II, sugerindo ação pró-oxidante do interferon e/ou da ribavirina. Após a suplementação antioxidante, observou-se diminuição das atividades enzimáticas, da CAT, GST e GR e vitamina E, além dos níveis de zinco e ferro em pacientes do grupo II. Por outro lado, a suplementação antioxidante no grupo III permitiu melhorar o poder redutor destes pacientes, aumentar os níveis de GSH, provavelmente a expensas da vitamina E, a qual apresentou níveis diminuídos, assim como os níveis de PC, ferro e zinco. Em suma, pacientes com VHC sem tratamento são passíveis de dano oxidativo enquanto que pacientes tratados com interferon e/ou ribavirina aparentemente apresentaram dano oxidativos aumentados e observou-se que suplementação antioxidante melhorou esta condição

Dano ao DNA, citotoxicidade, efeito antiproliferativo e antitumoral de 1,4-naftoquinonas substituídas

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Bioquímica, Florianópolis, 2014.As quinonas são uma classe de substâncias químicas de interesse na terapêutica do câncer, visto que algumas delas apresentam potencial antitumoral. Pesquisas sugerem que o efeito antitumoral destes compostos pode ser potenciado por substituições estratégicas dos substituintes ligados ao núcleo quinóide. Desta forma, este trabalho teve como objetivo caracterizar o potencial citotóxico, antiproliferativo e antitumoral bem como o mecanismo molecular de ação de 1,4-naftoquinonas substituídas. Dentre os compostos avaliados, os que apresentaram maior atividade citotóxica para linhagem tumoral MCF7 (câncer de mama humano), avaliada pelo método do MTT, foram DPB1, DPB2, DPB4 e DPB5 (CI50 Abstract : Quinones are a class of chemicals of interest in cancer therapy, since some of them have antitumor potential. Research suggests that the antitumor effect of these compounds can be potentiated by strategic substitutions quinoid substituents attached to the core. Thus, this study aimed to characterize the cytotoxic, antiproliferative and antitumor potential as well as the molecular mechanism of action of substituted 1,4- naphthoquinones. Among the compounds evaluated, showed the highest cytotoxic activity to tumor cell line MCF7 (human breast cancer), as evaluated by the MTT method were DPB1, DPB2, DPB4 and DPB5 (IC50 < 25 mM) having substituents attached directly to the naphthoquinone nucleus. Suggesting that the cytotoxic effect is mainly influenced by their substituents groups where donors and electron acceptors modulate the electronic properties of this nitrogen atom in molecules, facilitating or hindering the occurrence of the redox cycle. Furthermore, these same four compounds inhibited the formation of cell colonies by promoting a cytostatic effect. Through the fragmentation test and the plasmid DNA comet assay was found that 1,4- naphthoquinones substituted promoted direct DNA damage and that the photoactivation by UV-A light (365 nm) potentiated this damage. To verify whether they had direct interaction with DNA, the technique of scanning spectrometer was used, using the CT-DNA. It was observed that the compounds interact with the DNA, providing a hypochromic effect, indicating that the interaction is by intercalation, which was confirmed by fluorescence assay using ethidium bromide as the intercalating agent. The DNA damage induced by the compounds was also confirmed by immunoblot, substituted naphthoquinones increased phosphorylation of histone ?H2Ax. Furthermore, flow cytometry showed that the compounds evaluated promoted cell cycle arrest in the G2 phase, which was also confirmed by immunoelectrophoresis assay where no inhibition was observed in the expression of the cdk2 protein, which could be due to the fact this cyclin dependent kinase to be particularly involved in the regulation of the cell cycle in the G1 phase. Moreover, it was observed that the compounds promoted cell death predominantly by caspase -independent apoptosis and / or programmed necrosis both in vivo and in vitro. Whereas these compounds increased the expression of p53 and did not promote the cleavage of PARP. From these results, it is suggested that the compounds may have promoted cell death by necrosis or programmed like necrosis. It is also suggested that the cytotoxic, genotoxic and cytostatic effects promoted by the compounds are due to generation of ROS, which was observed by DCFH-A assay, where DPB1 , DPB2 , DPB4 and DPB5 compounds showed a significant increase in ROS generation (7874.22, 6056.37, 3833.33 and 10547.47 fluorescence units / mg protein ) compared to negative control (1493.10 fluorescence units/mg protein). In vivo assays showed that DPB1, DPB2, DPB4 and DPB5 showed antitumor effect, highlighting the compound DPB4. Regarding the type of cell death was observed that compounds increased the percentage of early apoptotic cells and mostly late and/or necrotic, confirming the results shown in vitro. Furthermore, we investigated whether the compounds could also have a antiangiogenic activity, this effect being observed for all compounds evaluated (DPB1, DPB2, DPB4 and DPB5), highlighting the compound DPB2 that at concentrations of 5, 10 and 20µM /ml reduced the percentage of vessels in 67.69, 53.84 and 76.92 %. Finally, it is suggested that the effects mediated by substituted 1,4- naphthoquinones study are due to the synergism of three mechanisms: redox cycle by the increased generation of ROS, and DNA intercalating / or inhibition of topoisomerase II, which consequently leads to tumor cell death

Antioxidant therapy attenuates oxidative insult caused by benzonidazole in chronic Chagas` heart disease

Chronic chagasic cardiac patients are exposed to oxidative stress that apparently contributes to disease progression. Benznidazole (BZN) is the main drug used for the treatment of chagasic patients and its action involves the generation of reactive species. 41 patients with Chagas` heart disease were selected and biomarkers of oxidative stress were measured before and after 2 months of BZN treatment (5 mg/kg/day) and the subsequent antioxidant supplementation with vitamin E (800 UI/day) and C (500 mg/day) during 6 months. Patients were classified according to the modified Los Andes clinical hemodynamic classification in groups IA, IB, II and III, and the activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione S-transferase (GST) and glutathione reductase (GR), as well as the contents of reduced glutathione (GSH), thiobarbituric acid reactive species (TBARS), protein carbonyl (PC), vitamin E and C and nitric oxide (NO), myeloperoxidase (MPO) and adenosine deaminase (ADA) activities were measured in their blood. Excepting in group III, after BZN treatment SOD, CAT, GPx and GST activities as well as PC levels were enhanced while vitamin E levels were decreased in these groups. After antioxidant supplementation the activities of SOD, GPx and GR were decreased whereas PC, TBARS, NO, and GSH levels were decreased. In conclusion, BZN treatment promoted an oxidative insult in such patients while the antioxidant supplementation was able to attenuate this effect by increasing vitamin E levels, decreasing PC and TBARS levels, inhibiting SOD, GPx and GR activities as well as inflammatory markers, mainly in stages with less cardiac involvement. (C) 2009 Elsevier Ireland Ltd. All rights reserved.CNPq (Conselho Nacional de Desenvolvimento Cientifico e Tecnologico)/MCT/Ministério da Saúde MS-SCTIE-DECITCNPq (Conselho Nacional de Desenvolvimento Cientifico e Tecnologico)/MCT/Ministério da Saúde MS-SCTIE-DECIT[409266/2006-0]CNPq[305018/2006-0]Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)PIBIC-CNP

Substituted 3‑acyl‑2‑phenylamino‑1,4‑naphthoquinones intercalate into DNA and cause genotoxicity through the increased generation of reactive oxygen species culminating in cell death

Naphthoquinones interact with biological systems by generating reactive oxygen species (ROS) that can damage cancer cells. The cytotoxicity and the antitumor activity of 3‑acyl‑2‑phenylamino‑1,4‑naphthoquinones (DPB1‑DPB9) were evaluated in the MCF7 human breast cancer cell line and in male Ehrlich tumor‑bearing Balb/c mice. DPB4 was the most cytotoxic derivative against MCF7 cells (EC50 15 µM) and DPB6 was the least cytotoxic one (EC50 56 µM). The 1,4‑naphthoquinone derivatives were able to cause DNA damage and promote DNA fragmentation as shown by the plasmid DNA cleavage assay (FII form). In addition, 1,4‑naphthoquinone derivatives possibly interacted with DNA as intercalating agents, which was demonstrated by the changes caused in the fluorescence of the DNA‑ethidium bromide complexes. Cell death of MCF7 cells induced by 3‑acyl‑2‑phenylamino‑1,4‑naphthoquinones was mostly due to apoptosis. The DNA fragmentation and subsequent apoptosis may be correlated to the redox potential of the 1,4‑naphthoquinone derivatives that, once present in the cell nucleus, led to the increased generation of ROS. Finally, certain 1,4‑naphthoquinone derivatives and particularly DPB4 significantly inhibited the growth of Ehrlich ascites tumors in mice (73%)

Sodium orthovanadate associated with pharmacological doses of ascorbate causes an increased generation of ROS in tumor cells that inhibits proliferation and triggers apoptosis

Pharmacological doses of ascorbate were evaluated for its ability to potentiate the toxicity of sodium orthovanadate (Na(3)VO(4)) in tumor cells. Cytotoxicity, inhibition of cell proliferation, generation of ROS and DNA fragmentation were assessed in T24 cells. Na(3)VO(4) was cytotoxic against T24 cells (EC(50)=5.8 μM at 24 h), but in the presence of ascorbate (100 μM) the EC(50) fell to 3.3 μM. Na(3)VO(4) plus ascorbate caused a strong inhibition of cell proliferation (up to 20%) and increased the generation of ROS (4-fold). Na(3)VO(4) did not directly cleave plasmid DNA, at this aspect no synergism was found occurring between Na(3)VO(4) and ascorbate once the resulting action of the combination was no greater than that of both substances administered separately. Cells from Ehrlich ascites carcinoma-bearing mice were used to determine the activity of antioxidant enzymes, the extent of the oxidative damage and the type of cell death. Na(3)VO(4) alone, or combined with ascorbate, increased catalase activity, but only Na(3)VO(4) plus ascorbate increased superoxide dismutase activity (up to 4-fold). Oxidative damage on proteins and lipids was higher due to the treatment done with Na(3)VO(4) plus ascorbate (2-3-fold). Ascorbate potentiated apoptosis in tumor cells from mice treated with Na(3)VO(4). The results indicate that pharmacological doses of ascorbate enhance the generation of ROS induced by Na(3)VO(4) in tumor cells causing inhibition of proliferation and apoptosis. Apoptosis induced by orthovanadate and ascorbate is closer related to inhibition on Bcl-xL and activation of Bax. Our data apparently rule out a mechanism of cell demise p53-dependent or related to Cdk2 impairment