In vitro basal cytotoxicity assay applied to estimate acute oral systemic toxicity of grandisin and its major metabolite

Preclinical investigations can start with preliminary in vitro studies before using animal models. Following this approach, the number of animals used in preclinical acute toxicity testing can be reduced. In this study, we employed an in-house validated in vitro cytotoxicity test based on the Spielmann approach for toxicity evaluation of the lignan grandisin, a candidate anticancer agent, and its major metabolite. the 4-O-demethylgrandisin, by neutral red uptake (NRU) assay, on mouse fibroblasts Balb/c 3T3 cell line. Using different concentrations of grandisin and its major metabolite (2.31; 1.16; 0.58; 0.29; 0.14; 0.07; 0.04; 0.002 mu M) in Balb/c 3T3-A31 NRU cytotoxicity assay, after incubation for 48 h, we obtained IC(50) values for grandisin and its metabolite of 0.078 and 0.043 mu M, respectively. The computed LD(50) of grandisin and 4-O-demethylgrandisin were 617.72 and 429.95 mg/kg, respectively. Both were classified under the Globally Harmonized System as category 4. Since pharmacological and toxicological data are crucial in the developmental stages of drug discovery, using an in vitro assay we demonstrated that grandisin and its metabolite exhibit distinct toxicity profiles. Furthermore, results presented in this work can contribute to reduce the number of animals required in subsequent pharmacological/toxicological studies. (C) 2010 Elsevier GmbH. All rights reserved.

Subcutaneous tissue reaction and cytotoxicity of polyvinylidene fluoride and polyvinylidene fluoride-trifluoroethylene blends associated with natural polymers

Cytotoxicity and subcutaneous tissue reaction of innovative blends composed by polyvinylidene fluoride and polyvinylidene fluoride-trifluoroethylene associated with natural polymers (natural rubber and native starch) forming membranes were evaluated, aiming its applications associated with bone regeneration. Cytotoxicity was evaluated in mouse fibroblasts culture cells (NIH3T3) using trypan blue staining. Tissue response was in vivo evaluated by subcutaneous implantation of materials in rats, taking into account the presence of necrosis and connective tissue capsule around implanted materials after 7, 14, 21, 28, 35, 60, and 100 days of surgery. The pattern of inflammation was evaluated by histomorphometry of the inflammatory cells. Chemical and morphological changes of implanted materials after 60 and 100 days were evaluated by Fourier transform infrared (FTIR) absorption spectroscopy and scanning electron microscopy (SEM) images. Cytotoxicity tests indicated a good tolerance of the cells to the biomaterial. The in vivo tissue response of all studied materials showed normal inflammatory pattern, characterized by a reduction of polymorphonuclear leukocytes and an increase in mononuclear leukocytes over the time (p < 0.05 Kruskal-Wallis). On day 60, microscopic analysis showed regression of the chronic inflammatory process around all materials. FTIR showed no changes in chemical composition of materials due to implantation, whereas SEM demonstrated the delivery of starch in the medium. Therefore, the results of the tests performed in vitro and in vivo show that the innovative blends can further be used as biomaterials. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 101B: 1284-1293, 2013. Copyright © 2013 Wiley Periodicals, Inc.

Silk Fibroin And Sodium Alginate Blend: Miscibility And Physical Characteristics.

Films of silk fibroin (SF) and sodium alginate (SA) blends were prepared by solution casting technique. The miscibility of SF and SA in those blends was evaluated and scanning electron microscopy (SEM) revealed that SF/SA 25/75 wt.% blends underwent microscopic phase separation, resulting in globular structures composed mainly of SF. X-ray diffraction indicated the amorphous nature of these blends, even after a treatment with ethanol that turned them insoluble in water. Thermal analyses of blends showed the peaks of degradation of pristine SF and SA shifted to intermediate temperatures. Water vapor permeability, swelling capacity and tensile strength of SF films could be enhanced by blending with SA. Cell viability remained between 90 and 100%, as indicated by in vitro cytotoxicity test. The SF/SA blend with self-assembled SF globules can be used to modulate structural and mechanical properties of the final material and may be used in designing high performance wound dressing.

Interaction of rheumatoid factor and Entamoeba histolytica

The amoebae's cytotoxicity test and the amoebae's lysis test were used to show possible interactions between rheumatoid factor (RF) and Entamoeba histolytica. Amoebae's cytotoxic activity (ACA) was inhibited by affinity chromatography purified antiamoebae rabbit IgG (RIgG). Enhanced inhibition could be demonstrated with RIgG plus RF. But the same marked inhibition of ACA could be seen when replacing RF by heat inactivated normal human serum as a control. About 50% amoebae's lysis occurred when amoebae were brought together with native normal human serum (NNHS) as a source of complement. Amoebae's lysis increased to 60% when incubated with NHS plus human antiamoebae antibodies. No further augmentation could be obtained by the addition of RF. Using RIgG instead of human antibodies the lysis rate did not increase. Incubation of amoebae, NNHS, RIgG and RF even reduced amoebae's lysis. RF neither has an effect on ACA nor on complement mediated AL in vitro.

Antibody-dependent cell-mediated cytolysis of human colon carcinoma cells induced by specific antisera against carcinoembryonic antigen.

Antibody-dependent lymphocyte cytotoxicity against human colon carcinoma cells grown in vitro was demonstrated with rabbit anti-carcinoembryonic antigen (CEA) antisera and normal human lymphocytes. The same antisera produced no tumor cell lysis in a complement-dependent cytotoxicity test. The specificity of the reaction was demonstrated by the inhibition of antibody-dependent lymphocyte cytotoxicity after the addition of increasing amounts of purified CEA to the antiserum and by the fact that only tumor cell lines expressing CEA on their surface were lysed. Antibody-dependent lymphocyte cytotoxicity was also observed against two colon carcinoma cell lines that expressed Blood Group A antigen, using a human serum containing anti-Blood Group A antibodies of the immunoglobulin G class. This reaction was specifically inhibited by absorption with Blood Group A red cells, whereas the anti-CEA-dependent cytotoxicity was not inhibited by absorption with red cells of different blood groups.

Association Behavior of Biotinylated and Non-Biotinylated PolyEthylene Oxide-b-Poly(2-(Diethylamino)Ethyl Methacrylate)

Biotinylated and non-biotinylated copolymers of ethylene oxide (EO) and 2-(diethylamino)ethyl methacrylate (DEAEMA) were synthesized by the atom transfer radical polymerization technique (ATRP). The chemical compositions of the copolymers as determined by NMR are represented by PEO₁₁₃PDEAEMA₇₀ and biotin-PEO₁₀₄PDEAEMA₉₃ respectively. The aggregation behavior of these polymers in aqueous solutions at different pHs and ionic strengths was studied using a combination of potentiometric titration, dynamic light scattering (DLS), static light scattering (SLS), and transmission electron microscopy (TEM). Both PEO-b-PDEAEMA and biotin-PEO-b-PDEAEMA diblock copolymers form micelles at high pH with hydrodynamic radii (Rh) of about 19 and 23 nm, respectively. At low pH, the copolymers are dispersed as unimers in solution with Rh of about 6-7 nm. However, at a physiological salt concentration (cs) of about 0.16M NaCl and a pH of 7-8, the copolymers form large loosely packed Guassian chains, which were not present at the low cs of 0.001M NaCl. The critical micelle concentrations (CMC) and the cytotoxicity of the copolymers were investigated to determine a suitable polymer concentration range for future biological applications. Both PEO-b-PDEAEMA and biotin-PEO-b-PDEAEMA diblock copolymers possess identical CMC values of about 0.0023 mg/g, while the cytotoxicity test indicated that the copolymers are not toxic up to 0.05mg/g (> 83% cell survival at this concentration).

Anti-Mycobacterium tuberculosis activity of fungus Phomopsis stipata

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

Antiviral activity of plants occurring in the state of minas gerais (Brazil): Part III

A total of 24 extracts from 14 plant species collected at the state of Minas Gerais, Brazil, and belonging to five botanical families (Annonaceae, Apocynaceae, Ochnaceae, Polygonaceae and Vitaceae) was screened for cytotoxicity in cultured Vero cells and for antiviral activity against human herpes virus type 1 (HSV-1), vaccinia virus (VACV) and murine encephalomyocarditis virus (EMCV). The highest cytotoxicity (CC 50 < 10 μg/mL) was observed for the ethanol extracts from Annona coriacea fruits and seeds. Extracts from Hancornia speciosa, Ouratea castaneafolia and O. semisrrata were the only ones that have shown activity against all the three viruses assayed. Extracts from Polygonum spectabile, Hancornia speciosa, Himatanthus phagedaenica, Ouratea spectabilis and O. semiserrata were the most active against HSV-1 (EC 50 < 50 mg/mL), with favorable SI values (8.0 to 10.0). Hancornia speciosa and Anaxagorea dolichocarpa were the most active against EMCV (EC 50 50 - 100 μg/mL), with reasonable SI values (5.2 to 6.1), while moderate to low activity (EC 50 > 100 μg/mL) was observed for Ouratea spectabilis and O. semiserrata. A total of 7 plant species, Ouratea semiserrata, O. spectabilis, O. castanaeafolia, Rollinia laurifolia, Cissus erosa, Polygonum spectabile, and Hancornia speciosa, were active against VACV, disclosing EC50 < 50 μg/mL and SI values ranging from 6.6 to 67.3. In total, 10 out of the 14 species were selected from a literature survey on plants used to treat viral diseases in Brazil; these species were responsible for 70% of the positive results.

Bothrops leucurus venom induces nephrotoxicity in the isolated perfused kidney and cultured renal tubular epithelia

Bites from snake (Bothrops genus) cause local tissue damage and systemic complications, which include alterations such as hemostatic system and acute renal failure (ARF). Recent studies suggest that ARF pathogenesis in snakebite envenomation is multifactorial and involves hemodynamic disturbances, immunologic reactions and direct nephrotoxicity. The aim of the work was to investigate the effects of the Bothrops leucurus venom (BlV) in the renal perfusion system and in cultured renal tubular cells of the type MDCK (Madin-Darby Canine kidney). BlV (10 μg/mL) reduced the perfusion pressure at 90 and 120 min. The renal vascular resistance (RVR) decreased at 120 min of perfusion. The effect on urinary flow (UF) and glomerular filtration rate (GFR) started 30 min after BlV infusion, was transient and returned to normal at 120 min of perfusion. It was also observed a decrease on percentual tubular transport of sodium (%TNa+) at 120 min and of chloride (%TCl-) at 60 and 90 min. The treatment with BlV caused decrease in cell viability to the lowest concentration tested with an IC50 of 1.25 μg/mL. Flow cytometry with annexin V and propidium iodide showed that cell death occurred predominantly by necrosis. However, a cell death process may involve apoptosis in lower concentrations. BlV treatment (1.25 μg/mL) led to significant depolarization of the mitochondrial membrane potential and, indeed, we found an increase in the expression of cell death genes in the lower concentrations tested. The venom also evoked an increase in the cytosolic Ca2+ in a concentration dependent manner, indicating that Ca2+ may participate in the venom of B. leucurus effect. The characterization of the effects in the isolated kidney and renal tubular cells gives strong evidences that the acute renal failure induced by this venom is a result of the direct nephrotoxicity which may involve the cell death mechanism. © 2012.

Evaluation of the effects of nicorandil and its molecular precursor (without radical NO) on proliferation and apoptosis of 786-cell

Nicorandil is a nitric oxide (NO) donor used in the treatment of angina symptoms. It has also been reported to protect cells and affect the proliferation and death of cells in some tissues. The molecules that interfere with these processes can cause dysfunction in healthy tissues but can also assist in the therapy of some disorders. In this study we examined the effect of nicorandil and of the molecular precursor that does not have the NO radical (N-(beta-hydroxyethyl) nicotinamide) on the cell proliferation and death of human renal carcinoma cells (786-O) under normal oxygenation conditions. The molecular precursor was used in order to analyze the effects independents of NO. In the cytotoxicity test, nicorandil was shown to be cytotoxic at very high concentrations and it was more cytotoxic than its precursor (cytotoxic at concentrations of 2,000 and 3,000 μg/mL, respectively). We propose that the lower cytotoxicity of the precursor is due to the absence of the NO radical. In this study, the cells exposed to nicorandil showed neither statistically significant changes in cell proliferation nor increases in apoptosis or genotoxicity. The precursor generated similar results to those of nicorandil. We conclude that nicorandil causes no changes in the proliferation or apoptosis of the cell 786-O in normal oxygenation conditions. Moreover, the lack of NO radical in the precursor molecule did not show a different result, except in the cell cytotoxicity. © 2013 Springer Science+Business Media Dordrecht.

Avaliação de apoptose induzida por extrato orgânico de material particulado atmosférico, rico em hidrocarbonetos policíclicos aromáticos, na região urbana de Araraquara durante a safra e entressafra de cana-de-açúcar

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Citotoxicidade e expressão gênica de fibrobastos pulpares tratados com extratos de monômeros resinosos e derivados vegetais

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

Prospecção química e avaliação de atividade biológica de Pothomorphe Umbellata frente a algumas linhagens de dermatófitos

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Avaliação da capacidade de mineralização e citotoxicidade do MTA, Sealapex e Sealapex plus

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)