Genotoxicity of the medicinal plant Maytenus robusta in mammalian cells in vivo.

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effects of neem oil (Azadirachta indica A. Juss) on midgut cells of predatory larvae Ceraeochrysa claveri (Navás, 1911) (Neuroptera: Chrysopidae)

The effects of ingested neem oil, a botanical insecticide obtained from the seeds of the neem tree, Azadirachta indica, on the midgut cells of predatory larvae Ceraeochrysa claveri were analyzed. C. claveri were fed on eggs of Diatraea saccharalis treated with neem oil at a concentration of 0.5%, 1% and 2% during throughout the larval period. Light and electron microscopy showed severe damages in columnar cells, which had many cytoplasmic protrusions, clustering and ruptured of the microvilli, swollen cells, ruptured cells, dilatation and vesiculation of rough endoplasmic reticulum, development of smooth endoplasmic reticulum, enlargement of extracellular spaces of the basal labyrinth, intercellular spaces and necrosis. The indirect ingestion of neem oil with prey can result in severe alterations showing direct cytotoxic effects of neem oil on midgut cells of C. claveri larvae. Therefore, the safety of neem oil to non-target species as larvae of C. claveri was refuted, thus the notion that plants derived are safer to non-target species must be questioned in future ecotoxicological studies. © 2012 Elsevier Ltd.

In vitro assessment of the cytotoxic, apoptotic, and mutagenic potentials of Isatin

Isatin (1H-indole-2,3-dione) is a chemical found in various medicinal plant species and responsible for a broad spectrum of pharmacological and biological properties that may be beneficial to human health, as an anticonvulsant, antibacterial, antifungal, antiviral, and anticancer agent. The aim of the present study was to determine in vitro the cytotoxic, mutagenic, and apoptotic effects of isatin on CHO-K1 and HeLa cells using the MTT viability assay (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide), micronucleus (MN) test, apoptosis index, and nuclear division index (NDI). The 5 isatin concentrations evaluated in the mutagenicity and apoptosis tests were 0.5, 1, 5, 10, and 50 μM, selected through a preliminary MTT assay. Positive (doxorubicin, DXR) and negative (phosphate buffered saline, PBS) control groups were also included in the analysis. Isatin did not exert a mutagenic effect on CHO-K1 after 3 and 24 h of treatment or on HeLa cells after 24 h. However, 10 and 50 μM concentrations inhibited cell proliferation and promoted apoptosis in both CHO-K1 and HeLa cells. Data indicate that the cytotoxic, apoptotic, and antiproliferative effects of isatin were concentration independent and cell line independent. The authors thank Profa Dra Eiko Nakagawa Itano for the use the spectrophotometer and the Conselho Nacional para o Desenvolvimento Científico e Tecnológico for master's scholarships to P. M. Cândido-Bacani and grants to T. R. Calvo, W. Vilegas, E. A. Varanda and I. M. S. Cólus. The Conselho Nacional para o Desenvolvimento Científico e Tecnológico provided funding for this study. © 2013 Taylor & Francis Group, LLC.

Antioxidant and cytotoxic effects of crude extract, fractions and 4-nerolidylcathecol from aerial parts of pothomorphe umbellata L. (Piperaceae)

The crude ethanolic extract from aerial parts of Pothomorphe umbellata L. (Piperaceae) and fractions obtained by partitions sequentially among water-methanol, methylene chloride, and ethyl acetate, as well as the major constituent, 4-nerolidylcatechol, were, respectively, evaluated and evidenced for antioxidant and cytotoxic effects through fluorometric microplate and microculture tetrazolium assays in HL-60 cells. The crude ethanolic extract demonstrated the preeminent antioxidant activity (IC50 = 1.2 μg/mL) against exogenous cytoplasmic reactive oxygen species, followed by the water-methanolic (IC50 = 4.5 μg/mL), methylene chloride (IC 50 = 5.9 g/mL), ethyl acetate (IC50 = 8.0 g/mL), 4-nerolidylcatechol (IC50 = 8.6 g/mL), and the sterol fractions (IC50 > 12.5 μg/mL). Vitamin C, the positive control used in this assay, presented IC50 value equivalent to 1.7 μg/mL. 4-Nerolidylcatechol (IC50 = 0.4 μg/mL) and methylene chloride fraction (IC50 = 2.3 μg/mL) presented considerable cytotoxicity probably because of the presence of an o-quinone, an auto-oxidation by product of the catechol. Polar compounds, present in the ethanol extract, appear to increase the solubility and stability of the major active constituent, acting synergistically with 4-nerolidylcatechol, improving its pharmacokinetic parameters and increasing significantly its antioxidant activity which, in turn, suggests that the aqueous-ethanolic extract, used in folklore medicine, is safe and effective. © 2013 Andrey P. Lopes et al.

Effects of zoledronic acid on odontoblast-like cells

The aim of the study was to evaluate the effects of a highly potent bisphosphonate, zoledronic acid (ZOL), on cultured odontoblast-like cells MDPC-23. The cells (1.5 × 104 cells/cm2) were seeded for 48 h in wells of 24-well dished. Then, the plain culture medium (DMEM) was replaced by fresh medium without fetal bovine serum. After 24 h, ZOL (1 or 5 μM) was added to the medium and maintained in contact with the cells for 24 h. After this period, the succinic dehydrogenase (SDH) enzyme production (cell viability-MTT assay), total protein (TP) production, alkaline phosphatase (ALP) activity, and gene expression (qPCR) of collagen type I (Col-I) and ALP were evaluated. Cell morphology was assessed by SEM. Five μM ZOL caused a significant decrease in SDH production. Both ZOL concentrations caused a dose-dependent significant decrease in TP production and ALP activity. ZOL also produced discret morphological alterations in the MDPC-23 cells. Regarding gene expression, 1 μM ZOL caused a significant increase in Col-I expression. Although 5 μM ZOL did not affect Col-I expression, it caused a significant alteration in ALP expression (ANOVA and Tukey's test, p < 0.05). ZOL presented a dose-dependent cytotoxic effect on the odontoblast-like cells, suggesting that under clinical conditions the release of this drug from dentin could cause damage to the pulpo-dentin complex. © 2012 Elsevier Ltd.

Nasal administration of liquid crystal precursor mucoadhesive vehicle as an alternative antiretroviral therapy

The purpose of this study was to develop a mucoadhesive stimuli-sensitive drug delivery system for nasal administration of zidovudine (AZT). The system was prepared by formulating a low viscosity precursor of a liquid crystal phase, taking advantage of its lyotropic phase behavior. Flow rheology measurements showed that the formulation composed of PPG-5-CETETH-20, oleic acid and water (55, 30, 15% w/w), denominated P, has Newtonian flow behavior. Polarized light microscopy (PLM) revealed that formulation P is isotropic, whereas its 1:1 (w/w) dilution with artificial nasal mucus (ANM) changed the system to an anisotropic lamellar phase (PD). Oscillatory frequency sweep analysis showed that PD has a high storage modulus (G′) at nasal temperatures. Measurement of the mucoadhesive force against excised porcine nasal mucosa or a mucin disk proved that the transition to the lamellar phase tripled the work of mucoadhesion. Ex vivo permeation studies across porcine nasal mucosa exhibited an 18-fold rise in the permeability of AZT from the formulation. The Weibull mathematical model suggested that the AZT is released by Fickian diffusion mechanisms. Hence, the physicochemical characterization, combined with ex vivo studies, revealed that the PPG-5-CETETH-20, oleic acid, and water formulation could form a mucoadhesive matrix in contact with nasal mucus that promoted nasal absorption of the AZT. For an in vivo assessment, the plasma concentrations of AZT in rats were determined by HPLC method following intravenous and intranasal administration of AZT-loaded P formulation (PA) and AZT solution, respectively, at a dose of 8 mg/kg. The intranasal administration of PA resulted in a fast absorption process (Tmax = 6.7 min). Therefore, a liquid crystal precursor formulation administered by the nasal route might represent a promising novel tool for the systemic delivery of AZT and other antiretroviral drugs. In the present study, the uptake of AZT absorption in the nasal mucosa was demonstrated, providing new foundations for clinical trials in patients with AIDS. © 2012 Elsevier B.V. All rights reserved.

Anticancer effects of geopropolis produced by stingless bees on canine osteosarcoma cells in vitro

Geopropolis is produced by indigenous stingless bees from the resinous material of plants, adding soil or clay. Its biological properties have not been investigated, such as propolis, and herein its cytotoxic action on canine osteosarcoma (OSA) cells was evaluated. OSA is a primary bone neoplasm diagnosed in dogs being an excellent model in vivo to study human OSA. spOS-2 primary cultures were isolated from the tumor of a dog with osteosarcoma and incubated with geopropolis, 70% ethanol (geopropolis solvent), and carboplatin after 6, 24, 48, and 72 hours. Cell viability was analyzed by the crystal violet method. Geopropolis was efficient against canine OSA cells in a dose- and time-dependent way, leading to a distinct morphology compared to control. Geopropolis cytotoxic action was exclusively due to its constituents since 70% ethanol (its solvent) had no effect on cell viability. Carboplatin had no effect on OSA cells. Geopropolis exerted a cytotoxic effect on canine osteosarcoma, and its introduction as a possible therapeutic agent in vivo could be investigated, providing a new contribution to OSA treatment. © 2013 Naiara Costa Cinegaglia et al.

Toxic effects of daily applications of 10% carbamide peroxide on odontoblast-like MDPC-23 cells

Background. Tooth bleaching has been widely studied, mainly due to the possible undesirable effects that can be caused by this esthetic procedure. The cytotoxicity of the bleaching agents and its components to pulp cells has been demonstrated in several researches. The aim of this study was to evaluate the toxic effects of successive applications of 10% carbamide peroxide (CP) gel on odontoblast-like cells. Materials and methods. Enamel-dentin discs obtained from bovine incisors were adapted to artificial pulp chambers (APCs). The groups were formed as follows: G1: Without treatment (control group); G2: 10% carbamide peroxide, CP (five applications/one per day); G3: 10% CP (one unique application); and G4: 35% hydrogen peroxide, HP (three applications of 15 min each). After treatment, cell metabolism (MTT), alkaline phosphatase (ALP) activity and plasma membrane damage (flow cytometry) were analyzed. Results. Reductions in cell metabolism and alkaline phosphatase activity along with severe damage of the cytoplasmic membrane were noted in G2. In G3, no damage was observed, compared to the control group. Intermediary values of toxicity were obtained after 35% HP application. Conclusion. It can be concluded that one application of 10% CP did not cause toxic effects in odontoblast-like cells, but the successive application of this product promoted severe cytotoxic effects. The daily application of the bleaching agents, such as used in the at-home bleaching technique, can increase the damages caused by this treatment to the dental pulp cells. © 2013 Informa Healthcare.

Glutathione and glutathione peroxidase expression in breast cancer: An immunohistochemical and molecular study

The use of prognostic markers for breast cancer allows therapeutic strategies to be defined more efficiently. The expression of glutathione (GSH) and glutathione peroxidase (GPX) in tumor cells has been evaluated as a predictor of prognosis and response to cytotoxic treatments. Its immunoexpression was assessed in 63 women diagnosed with invasive ductal carcinoma in a retrospective study. The results showed that high GSH expression was associated with tumors negative for the estrogen receptor (ER) (P<0.05), and GPX expression was associated with tumors negative for the progesterone receptor (PR) and patient mortality. Focusing on the 37 patients who received adjuvant chemotherapy/radiotherapy (Group I), high expression of GPX was associated with a high rate of patient mortality (P<0.05). The 19 patients who received only adjuvant chemotherapy (Group II) showed high expression of GSH in relation to metastasis (P<0.05). In addition, high levels of GPX expression were significantly associated with a shorter overall survival (P<0.05). To confirm this, the expression of precursor genes of GSH [glutamate cysteine ligase (GCLC) and glutathione synthetase (GSS)] and the GPX gene was analyzed using quantitative PCR in cultured neoplastic mammary cells treated with doxorubicin. Doxorubicin treatment was able to eliminate tumor cells without alterations in the gene expression of GSS, but led to underexpression of the GCLC and GPX genes. Our results suggest that high levels of GPX may be related to the development of resistance to chemotherapy in these tumors, response to treatment and the clinical course of the breast cancer patients.

Cytotoxic, genotoxic/antigenotoxic and mutagenic/antimutagenic effects of the venom of the wasp Polybia paulista

Hymenoptera venoms are constituted by a complex mixture of chemically or pharmacologically bioactive agents, such as phospholipases, hyaluronidases and mastoparans. Venoms can also contain substances that are able to inhibit and/or diminish the genotoxic or mutagenic action of other compounds that are capable of promoting damages in the genetic material. Thus, the present study aimed to assess the effect of the venom of Polybia paulista, a neotropical wasp, by assays with HepG2 cells maintained in culture. The cytotoxic potential of the wasp venom, assessed by the methyl thiazolyl tetrazolium assay (MTT assay), was tested for the concentrations of 10μg/mL, 5μg/mL and 1μg/mL. As these concentrations were not cytotoxic, they were used to evaluate the genotoxic (comet assay) and mutagenic potential (micronucleus test) of the venom. In this study, it was verified that these concentrations induced damages in the DNA of the exposed cells, and it was necessary to test lower concentrations until it was found those that were not considered genotoxic and mutagenic. The concentrations of 1ng/mL, 100pg/mL and 10pg/mL, which did not induce genotoxicity and mutagenicity, were used in four different treatments (post-treatment, pre-treatment, simultaneous treatment with and without incubation), in order to evaluate if these concentrations were able to inhibit or decrease the genotoxic and mutagenic action of methyl methanesulfonate (MMS). None of the concentrations was able to inhibit and/or decrease the MMS activity. The genotoxic and mutagenic activity of the venom of P. paulista could be caused by the action of phospholipase, mastoparan and hyaluronidase, which are able to disrupt the cell membrane and thereby interact with the genetic material of the cells or even facilitate the entrance of other compounds of the venom that can act on the DNA. Another possible explanation for the genotoxicity and mutagenicity of the venom can be the presence of substances able to trigger inflammatory process and, consequently, generate oxygen reactive species that can interact with the DNA of the exposed cells. © 2013 Elsevier Ltd.

Impact of Epstein-Barr virus load, virus genotype, and frequency of the 30bp deletion in the viral BNLF-1 gene in patients harboring the human immunodeficiency virus

Patients infected with the human immunodeficiency virus (HIV) are at higher risk of developing Epstein-Barr Virus (EBV)-associated lymphomas. The usefulness of monitoring EBV in peripheral blood mononuclear cells (PBMCs) of patients infected with HIV has not been established. The aim of this study was to evaluate the EBV viral load in PBMCs, the frequency of viral genotypes, and the presence of the 30-bp deletion in the BNLF-1 gene. DNA samples from 156 patients attending the HIV/AIDS Day Clinic at Botucatu School of Medicine, Sao Paulo State University were evaluated. The EBV viral load was detectable by real time PCR in 123/156 (78.8%) cases and was higher in patients not receiving antiretroviral treatment or under therapeutic failure than in patients under successful highly active antiretroviral therapy (HAART) (P=0.0076). Overall, the profile of patients with high EBV viral load included elevated HIV viremia (P=0.0005), longer time of HIV diagnosis (P=0.0026), and increased levels of T CD8 + lymphocytes (P=0.0159). The successful amplification of the EBNA-2 gene by nested-PCR was achieved in 95 of 123 (77.2%) cases, of which 75.8% were EBV-1, 9.5% EBV-2, and 14.7% were co-infected with both EBV-1 and -2. The analysis of the BNLF-1 gene was possible in 99 of 123 (80.5%) cases, of which 50.5% had the 30-bp deletion. EBV-1 was more common than EBV-2, which may reflect the fact that the cohort was predominantly Caucasian and heterosexual. J. Med. Virol. 85:2110-2118, 2013. © 2013 Wiley Periodicals, Inc.

Avaliação da prliferação e apoptose com a utilização de arranjos em matriz de amostra tecidual (Tissue Microarray - TMA) em mastocitomas cutâneos caninos

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Estresse oxidativo em doentes falciformes: influência dos haplótipos e udo de medicação específica

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Síntese, caracterização e investigação das atividades biológicas de compostos ciclopalados

Pós-graduação em Química - IQ

Avaliação de mutagenicidade, citotoxicidade e expressão de proteínas relacionadas a apoptose, Bak, BCL-2 e P53 fosforilado, em células tratadas com fuligem e particulado total de queima de cana-de-açúcar

Pós-graduação em Biociências e Biotecnologia Aplicadas à Farmácia - FCFAR