Evaluation of extracts from Coccoloba mollis using the Salmonella/microsome system and in vivo tests

The common everyday use of medicinal plants is an ancient, and still very widespread practice, whereby the need for studies on their possible toxicity and mutagenic properties. The species Coccoloba mollis has been much used in phytotherapy, mainly in cases involving loss of memory and stress. In order to investigate its genotoxic and mutagenic potential, ethanolic extracts from the leaves and roots underwent Salmonella/microsome assaying (TA98 and TA100 strains, with and without exogenous metabolism – S9), besides comet and micronucleus tests in vivo.There was no significant increase in the number of revertants/plate of Salmonella strains in any of the analyzed root-extract concentrations, although the extract itself was extremely toxic to the Salmonella TA98 strain in the tests carried out with S9 (doses varying from 0.005 to 0.5 μg/plate). On the other hand, the leaf-extract induced mutations in the TA98 strain in the absence of S9 in the highest concentration evaluated, although at very low mutagenic potency (0.004 rev/ μg). Furthermore, there was no statistically significant increase in the number of comets and micronuclei, in treatments involving Swiss mice. It was obvious that extracts of Coccoloba mollis, under the described experimental conditions, are not mutagenic

Estudo do flavonóide rutina na citotoxicidade e análise de biomarcadores gênicos e bioquímicos de estresse genotóxico e oxidativo em cultura de células

Há um interesse crescente na investigação de substâncias de origem natural como os flavonóides, devido ao grande número de evidências dos benefícios que eles proporcionam para a saúde, principalmente reduzindo o desenvolvimento de doenças crônicas. Nesse contexto, a rutina é um flavonóide que tem sido bem caracterizado na literatura por apresentar uma variedade de atividades farmacológicas incluindo propriedades antiinflamatória, antialérgica, antimutagênica, imunomoduladora e hepatoprotetora. Estes efeitos protetores atribuídos a rutina estão relacionados, em grande parte, à sua propriedade antioxidante direta, no entanto, as vias moleculares e os mecanismos de ação desses processos são pouco conhecidos. Dessa forma, o objetivo do presente trabalho foi investigar os efeitos citotóxicos, genotóxicos e protetores do flavonóide rutina em concentrações fisiológicas relevantes in vtiro. Para isso, foram utilizadas quatro linhagens de células tumorais: HTC (hepatoma de Rattus norvegicus); HepG2 (hepatoma humano); HT-29 (adenocarcinoma de cólon humano) e 786-O (carcinoma renal humano). Os resultados obtidos com as células HTC mostraram que a concentração de 810 uM de rutina não foi citóxica, apresentou efeito genotóxico e não alterou a expressão dos genes GSTa2 e p38, relacionados ao metabolismo de drogas e controle do ciclo celular, respectivamente. Além disso, a análise dos espectros de Ressonância Magnética Nuclear (RMN H1) dos metábolitos das células expostas a concentração genotóxica da rutina (810uM), revelou menores níveis de fosfocolina, glicerofosfocolina, creatina, lactato, acetato e glutationa. A análise dos resultados das linhagens HepG2 e HT-29 revelou que 100 μM de rutina foi citotóxica e reduziu a expressão e a atividade das principais enzimas antioxidantes endógenas: superóxido dismutase...There is growing interest in the investigation of naturally occurring substances such as flavonoids, due to the large volume of evidence of the benefits they provide for health, mainly by reducing the development of chronic diseases. In this context, rutin is a flavonoid that has been well characterized in the literature by presenting a variety of pharmacological activities including antiinflammatory, antiallergic, antimutagenic, immunomodulating and hepatoprotective. These protective effects attributed to rutin are related in large part to their direct antioxidant properties, however, the molecular pathways and mechanisms of action of these processes are poorly understood. Thus, the objective of this study was to investigate the cytotoxic, genotoxic and protective flavonoid rutin in the relevant physiological concentrations in vtiro. Therefore, it was using four different strains: HTC (Rattus norvegicus hepatocellular); HepG2 (Human carcinoma), HT-29 (human colon adenocarcinoma) and 786- O (human kidney carcinoma). The results obtained with HTC cells showed that the concentration of 810 UM genotoxic effect of rutin showed no changes in gene expression GSTa2 and p38, related to drug metabolism and cell cycle control, respectively. In addition, NMR analysis of cells exposed to the genotoxic concentration rutin (810 UM) revealed lower levels of phosphocholine, glicerphosphocholine, creatine, lactate, acetate and glutathione. The results of the lines HepG2 and HT-29 revealed that 100 UM of rutin was cytotoxic and reduced the expression and activity of the major endogenous antioxidant enzymes: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (Gr). On the other hand, 10 UM of rutin did not alter the viability and kinetics of cell proliferation, significantly increased the... (Complete abstract click electronic access below

Effects of (-)-cubebin (Piper cubeba) on cytotoxicity, mutagenicity and expression of p38 MAP kinase and GSTa2 in a hepatoma cell line

(-)-Cubebin is a lignan extracted from the seeds of the pepper Piper cubeba, a commonly eaten spice with beneficial properties, including trypanocidal, anti-inflammatory, analgesic, anti-proliferative and leishmanicidal activities. Because of its therapeutic potential, we investigated the effects of (-)-cubebin on the cytotoxicity, cell proliferation kinetics, mutagenicity and expression of p38 MAP kinase and glutathione S-transferase a2 (GSTa2) using real-time RT-PCR in Rattus norvegicus hepatoma cells. We found that 280 μM (-)-cubebin was cytotoxic after 24, 48 and 72. h of exposure, but not mutagenic at 0.28 μM, 2.8 μM and 28 μM after 26. h. Similarly, exposure to 0.28 μM, 2.8 μM and 28 μM (-)-cubebin for 24, 48, 72 and 96. h did not alter the cell proliferation kinetics. Cells exposed to 28 μM (-)-cubebin for 24. h did not exhibit changes in p38 MAP kinase and GSTa2 expression, indicating that cellular changes were not induced by extracellular stimuli and that (-)-cubebin is likely not metabolized via this pathway. Our results suggest that high levels of (-)-cubebin should be consumed with caution due to the cytotoxic effect observed at the highest concentration. However, at lower concentrations, no cytotoxic, mutagenic or proliferative effects were observed, providing further evidence of the safety of consuming (-)-cubebin. © 2013 Elsevier Inc

Modulation of the mutagenic effect of benzo[a]pyrene and bleomycin by isoflavone extracts in a rat hepatoma cell line<br> Modulação do efeito mutagênico do benzo[a]pireno e bleomicina por extratos de isoflavonas em células de hepatoma de roedor

Epidemiologic studies show that the intake of foods rich in isoflavones (phytoestrogens), such as soybeans, confers protection against various types of cancer, what increases the scientific and popular interest on these compounds. In the present study, phytoestrogens extracts from soybeans were tested for genotoxic potential and modulatory effects on benzo[a]pyrene and bleomycin. Two phytoestrogens were evaluated in vitro, phytoestrogen “A” was supplied by EMBRAPA-Soja, Londrina – PR, and phytoestrogen “B” was purchased in a local drug store. The methods used were the comet assay (genotoxicity and antigenotoxicity) and micronucleus test with cytokinesis block (mutagenicity) in rat hepatoma cells (HTC cell). The isoflavones were tested at three concentrations pre-established by the MTT cytotoxicity assay. Both isoflavone extracts showed no genotoxic effects in the comet assay, but showed induction of micronucleus. In the evaluation of the phytoestrogens for a modulatory effect, both phytoestrogens extracts showed antigenotoxicity in the comet assay.Estudos epidemiológicos mostram que a ingestão de alimentos ricos em isoflavonas (fitoestrógenos), como a soja, confere proteção contra vários tipos de câncer, o que aumenta o interesse científico e popular sobre esses compostos. No presente estudo, os fitoestrógenos de extrato de soja foram testados quanto aos efeitos genotóxicos e modulador de benzo [a] pireno e bleomicina. Dois fitoestrogênios foram avaliados in vitro, o fitoestrógenos “A” foi fornecido pela Embrapa-Soja, Londrina - PR, e o fitoestrógenos “B” foi comprado em uma farmácia de manipulação local. Os métodos utilizados foram o teste do Cometa (genotoxicidade e antigenotoxicidade) e teste do Micronúcleo com Bloqueio Citocinese (mutagenicidade) em células de hepatoma de rato (HTC celulares). As isoflavonas foram testadas em três concentrações pré-estabelecidas pelo ensaio de citotoxidade MTT. Ambos os extratos de isoflavonas não mostraram efeitos genotóxicos no ensaio do cometa, mas mostraram indução de micronúcleo. Na avaliação dos fitoestrogênios para um efeito modulador, ambos os extratos fitoestrogênios mostraram efeito antigenotóxico no ensaio do cometa

Changes in gene expression in PBMCs profiles of PPAR alpha target genes in obese and non-obese individuals during fasting

Background: The prevalence of obesity has risen dramatically and the World Health Organization estimates that 700 million people will be obese worldwide by 2015. Approximately, 50% of the Brazilian population above 20 years of age is overweight, and 16% is obese. Aim: This study aimed to evaluate the differences in the expression of PPAR alpha target genes in human peripheral blood mononuclear cells (PBMCs) and free fatty acids (FFA) in obese and non-obese individuals after 24 h of fasting. We first presented evidence that Brazilian people exhibit expression changes in PPARa target genes in PBMCs under fasting conditions. Methods: Q-PCR was utilized to assess the mRNA expression levels of target genes. Results: In both groups, the FFA concentrations in. creased significantly after 24 h of fasting. The basal FFA mean concentration was two-fold higher in the obese group compared with the non-obese group. After fasting, all genes evaluated in this study showed increased expression levels compared with basal expression in both groups. Conclusion: However, our results reveal no differences in gene expression between the obese and non-obese, more studies are necessary to precisely delineate the associated mechanisms, particularly those that include groups with different degrees of obesity and patients with diabetes mellitus type 2 because the expression of the main genes that are involved in beta-oxidation and glucose level maintenance are affected by these factors.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Changes in gene expression in PBMCs profiles of PPARα Target genes in obese and non-obese individuals during fasting

<p>Background: The prevalence of obesity has risen dramatically and the World Health Organization estimates that 700 million people will be obese worldwide by 2015. Approximately, 50% of the Brazilian population above 20 years of age is overweight, and 16% is obese. Aim: This study aimed to evaluate the differences in the expression of PPARα target genes in human peripheral blood mononuclear cells (PBMCs) and free fatty acids (FFA) in obese and non-obese individuals after 24 h of fasting. We first presented evidence that Brazilian people exhibit expression changes in PPARα target genes in PBMCs under fasting conditions. Methods: Q-PCR was utilized to assess the mRNA expression levels of target genes. Results: In both groups, the FFA concentrations increased significantly after 24 h of fasting. The basal FFA mean concentration was two-fold higher in the obese group compared with the non-obese group. After fasting, all genes evaluated in this study showed increased expression levels compared with basal expression in both groups. Conclusion: However, our results reveal no differences in gene expression between the obese and non-obese, more studies are necessary to precisely delineate the associated mechanisms, particularly those that include groups with different degrees of obesity and patients with diabetes mellitus type 2 because the expression of the main genes that are involved in β-oxidation and glucose level maintenance are affected by these factors.</p

Investigation of cytotoxic, apoptosis-inducing, genotoxic and protective effects of the flavonoid rutin in HTC hepatic cells

Rutin is a flavonoid with antioxidant, vasodilatory, anti-inflammatory and immune-stimulating activities. To study the toxicity of rutin and its protective effect, this work investigated the cytotoxic, apoptosis-inducing, genotoxic and protective effects of rutin in HTC cells. In the MTT assay, the highest concentration tested (810 mu M) showed cytotoxicity after 72 h of treatment, where cell viability and cell proliferation was diminished. None of the concentrations of rutin tested induced apoptosis after 24 h treatment. The highest concentration of rutin after 24 h treatment induced DNA damage, shown in the comet assay, but did have a genotoxic effect in the micronucleus test. Rutin was tested against the pro-carcinogenic agent benzo(a)pyrene, at concentrations of 90, 270 and 810 mu M, and was found to reduce induced DNA damage significantly. This protective effect of rutin against a pro-carcinogen, suggests an important biological activity for this compound, which can contribute to human health through the diet. (C) 2010 Elsevier GmbH. All rights reserved

The azo dyes Disperse Red 1 and Disperse Orange 1 increase the micronuclei frequencies in human lymphocytes and in HepG2 cells

The use of azo dyes by different industries can cause direct and/or indirect effects oil human and environmental health due to the discharge of industrial effluents that contain these toxic compounds. Several studies have demonstrated the genotoxic effects of various azo dyes, but information on the DNA damage caused by Disperse Red 1 and Disperse Orange 1 is unavailable, although these dyes are used in dyeing processes in many countries. The aim of the present study was to evaluate the mutagenic activity of Disperse Red 1 and Disperse Orange 1 using the micronucleus (MN) assay in human lymphocytes and in HepG2 cells. In the lymphocyte assay. it was found that the number of MN induced by the lowest concentration of each dye (0.2 mu g/mL) was similar to that of the negative control. At the other concentrations, a dose response MN formation was observed up to 1.0 mu g/mL. At higher dose levels, the number of MN decreased. For the HepG2 cells the results were similar. With both dyes a dose dependent increase in the frequency of MN was detected. However for the HepG2, the threshold for this increase was 2.0 mu g/mL, while at higher doses a reduction in the MN number was observed. The proliferation index was also calculated in order to evaluate acute toxicity during the test. No differences were detected between the different concentrations tested and the negative control. (C) 2009 Elsevier B.V. All rights reserved.Faculdade de Ciencias Farmaceuticas de Ribeirao PretoUniversidade de Sao Paulo (USP)FAPESPCAPE