In vitro photodynamic therapy on human oral keratinocytes using chloroaluminum-phthalocyanine

In this study, oral carcinoma cells were used to evaluate chloroaluminum-phthalocyanine encapsulated in liposomes as the photosensitizer agent in support of photodynamic therapy (PDT). The genotoxicity and cytotoxicity behavior of the encapsulated photosensitizer in both dark and under irradiation using the 670-nm laser were investigated with the classical trypan blue cell viability test, the acridine orange/ethidium bromide staining organelles test, micronucleus formation frequency, DNA fragmentation, and cell morphology. The cell morphology investigation was carried out using light and electronic microscopes. Our findings after PDT include reduction in cell viability (95%) associated with morphologic alterations. The neoplastic cell destruction was predominantly started by a necrotic process, according to the assay with acridine orange and ethidium bromide, and this was confirmed by electronic microscopy analysis. Neither the PDT agent nor laser irradiation alone showed cytotoxicity, genotoxicity, or even morphologic alterations. Our results reinforce the efficiency of tight-irradiated chloroaluminum-phthalocyanine in inducing a positive effect of PDT. (C) 2008 Elsevier Ltd. All rights reserved.

In vitro effect of Aloe vera, Coriandrum sativum and Ricinus communis fractions on Leishmania infantum and on murine monocytic cells

In South America, visceral leishmaniasis is a zoonosis caused by the protozoan species Leishmania infantum (syn. L. chagasi) and is primarily transmitted through the bite of the female Lutzomyia longipalpis. Its main reservoir in urban areas is the dog. The application of control measures recommended by health agencies have not achieved significant results in reducing the incidence of human cases, and the lack of effective drugs to treat dogs resulted in the prohibition of this course of action in Brazil. Therefore, it is necessary to search new alternatives for the treatment of canine and human visceral leishmaniasis. The objectives of this study were to evaluate the in vitro effect of fractions from Aloe vera (aloe), Coriandrum sativum (coriander), and Ricinus communis (castor) on promastigotes and amastigotes of L. infantum and to analyze the toxicity against the murine monocytic cells RAW 264.7. To determine the viability of these substances on 50% parasites (IC50), we used a tetrazolium dye (MU) colorimetric assay (bromide 3-4.5-dimethylthiazol-2-yl-2,5-dephenyltetrazolium), and on amastigotes we performed an in situ ELISA. All fractions were effective against L. infantum promastigotes and did not differ from the positive control pentamidine (p > 0.05). However, the R. communis ethyl acetate and chloroform fractions, as well as the C. sativum methanol fraction, were the most effective against amastigotes and did not differ from the positive control amphotericin B (p > 0.05). The R. communis ethyl acetate fraction was the least toxic, presenting 83.5% viability of RAW 264.7 cells, which was similar to the results obtained with amphotericin B (p > 0.05). Based on these results, we intend to undertake in vivo studies with R. communis ethyl acetate fractions due the high effectiveness against amastigotes and promastigotes of L. infantum and the low cytotoxicity towards murine monocytic cells. (C) 2011 Elsevier B.V. All rights reserved.

Antibody-targeted horseradish peroxidase associated with indole-3-acetic acid induces apoptosis in vitro in hematological malignancies

Indole-3-acetic acid (IAA), when oxidized by horseradish peroxidase (HRP), is transformed into cytotoxic molecules capable of inducing cell injury. The aim of this study was to test if, by targeting hematopoietic tumors with HRP-conjugated antibodies in association with IAA treatment, there is induction of apoptosis. We used two lineages of hematologic tumors: NB4, derived from acute promyelocytic leukemia (APL) and Granta-519 from mantle cell lymphoma (MCL). We also tested cells from 12 patients with acute myeloid leukemia (AML) and from 10 patients with chronic lymphocytic leukemia (CLL). HRP targeting was performed with anti-CD33 or anti-CD19 antibodies (depending on the origin of the cell), followed by incubation with goat anti-mouse antibody conjugated with HRP. Eight experimental groups were analyzed: control, HRP targeted, HRP targeted and incubated with 1, 5 and 10 mM IAA, and cells not HRP targeted but incubated with 1, 5 and 10 mM IAA. Apoptosis was analyzed by flow cytometry using annexin V-FITC and propidium iodide labeling. Results showed that apoptosis was dependent on the dose of IAA utilized, the duration of exposure to the prodrug and the origin of the neoplasia. Targeting HRP with antibodies was efficient in activating IAA and inducing apoptosis. (C) 2010 Elsevier Ltd. All rights reserved.

A reassessment of the in vitro RBC haemolysis assay with defibrinated sheep blood for the determination of the ocular irritation potential of cosmetic products: Comparison with the in vivo Draize rabbit test

We examined the correlation between results obtained from the in vivo Draize test for ocular irritation and in vitro results obtained from the sheep red blood cell (RBC) haemolytic assay, which assesses haemolysis and protein denaturation in erythrocytes, induced by cosmetic products. We sought to validate the haemolytic assay as a preliminary test for identifying highly-irritative products, and also to evaluate the in vitro test as alternative assay for replacement of the in vivo test. In vitro and in vivo analyses were carried out on 19 cosmetic products, in order to correlate the lesions in the ocular structures with three in vitro parameters: (i) the extent of haemolysis (H50); (ii) the protein denaturation index (131); and (iii) the H50/DI ratio, which reflects the irritation potential (IP). There was significant correlation between maximum average scores (MAS) and the parameters determined in vitro (r = 0.752-0.764). These results indicate that the RBC assay is a useful and rapid test for use as a screening method to assess the IP of cosmetic products, and for predicting the IP value with a high level of concordance (94.7%). The assay showed high sensitivity and specificity rates of 91.6% and 100%, respectively.

Mechanisms of Action of Eicosapentaenoic Acid in Bladder Cancer Cells In Vitro: Alterations in Mitochondrial Metabolism, Reactive Oxygen Species Generation and Apoptosis Induction

Purpose: Eicosapentaenoic acid has been tested in bladder cancer as a synergistic cytotoxic agent in the form of meglumine-eicosapentaenoic acid, although its mechanism of action is poorly understood in this cancer. The current study analyzed the mechanisms by which eicosapentaenoic acid alters T24/83 human bladder cancer metabolism in vitro. Materials and Methods: T24/83 human bladder cancer cells were exposed to eicosapentaenoic acid for 6 to 24 hours in vitro and incorporation profiles were determined. Effects on membrane phospholipid incorporation, energy metabolism, mitochondrial activity, cell proliferation and apoptosis were analyzed Reactive oxygen species and lipid peroxide production were also determined. Results: Eicosapentaenoic acid was readily incorporated into membrane phospholipids with a considerable amount present in mitochondrial cardiolipin. Energy metabolism was significantly altered by eicosapentaenoic acid, accompanied by decreased mitochondrial membrane potential, and increased lipid peroxide and reactive oxygen species generation. Subsequently caspase-3 activation and apoptosis were detected in eicosapentaenoic acid exposed cells, leading to decreased cell numbers. Conclusions: These findings confirm that eicosapentaenoic acid is a potent cytotoxic agent in bladder cancer cells and provide important insight into the mechanisms by which eicosapentaenoic acid causes these changes. The changes in membrane composition that can occur with eicosapentaenoic acid likely contribute to the enhanced drug cytotoxicity reported previously in meglumine-eicosapentaenoic acid/epirubicin/mitomycin studies. Dietary manipulation of the cardiolipin fatty acid composition may provide an additional method for stimulating cell death in bladder cancer. In vivo studies using intravesical and dietary manipulation of fatty acid metabolism in bladder cancer merit further attention.

Understanding the impact of divalent cation substitution on hydroxyapatite: An in vitro multiparametric study on biocompatibility

Hydroxyapatite (HA), a stable and biocompatible material for bone tissue therapy, may present a variable stoichiometry and accept a large number of cationic substitutions. Such substitutions may modify the chemical activity of HA surface, with possible impact on biocompatibility. In this work, we assessed the effects of calcium substitution with diverse divalent cations (Pb(2+), Sr(2+), Co(2+), Zn(2+), Fe(2+), Cu(2+), or Mg(2+)) on the biological behavior of HA. Physicochemical analyses revealed that apatite characteristics related to crystallinity and calcium dissolution/uptake rates are very sensitive to the nature of cationic substitution. Cytocompatibility was evaluated by mitochondrial activity, membrane integrity, cell density, proapoptotic potential, and adhesion tests. With the exception of Zn-HA, all the substituted HAs induced some level of apoptosis. The highest apoptosis levels were observed for Mg-HA and Co-HA. Cu-HA was the only material to impair simultaneously mitochondrial activity, membrane integrity, and cell density. The highest relative cell densities after exposure to the modified HAs were observed for Mg-HA and Zn-HA, while Co-HA significantly improved cell adhesion onto HA surface. These results show that changes on surface dissolution caused by cationic substitution, as well as the increase of metal species released to biological media, were the main responsible factors related to alterations on HA biocompatibility. (C) 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 98A: 351-358, 2011.

Violacein Extracted from Chromobacterium violaceum Inhibits Plasmodium Growth In Vitro and In Vivo

Violacein is a violet pigment extracted from the gram-negative bacterium Chromobacterium violaceum. It presents bactericidal, tumoricidal, trypanocidal, and antileishmanial activities. We show that micromolar concentrations efficiently killed chloroquine-sensitive and -resistant Plasmodium falciparum strains in vitro; inhibited parasitemia in vivo, even after parasite establishment; and protected Plasmodium chabaudi chabaudi-infected mice from a lethal challenge.

Synthesis, characterization, X-ray structure and in vitro anti mycobacterial and antitumoral activities of Ru(II) phosphine/diimine complexes containing the ""SpymMe(2)"" ligand, SpymMe(2)=4,6-dimethyl-2-mercaptopyrimidine

The reaction of cis-[RuCl2(dppb)(N-N)], dppb = 1,4-bis(diphenylphosphino)butane, complexes with the ligand HSpymMe(2), 4,6-dimethyl-2-mercaptopyrimidine, yielded the cationic complexes [Ru(SpymMe(2))(dppb)(N-N)]PF6, N-N = bipy (1) and Me-bipy (2), bipy = 2,2`-bipyridine and Me-bipy = 4,4`dimethyl-2,2`-bipyridine, which were characterized by spectroscopic and electrochemical techniques and X-ray crystallography and elemental analysis. Additionally, preliminary in vitro tests for antimycobacterial activity against Mycobacterium tuberculosis H37Rv ATCC 27264 and antitumor activity against the MDA-MB-231 human breast tumor cell line were carried out on the new complexes and also on the precursors cis-[RuCl2(dppb)(N-N)], N-N = bipy (3) and Me-bipy (4) and the free ligands dppb, bipy, Me-bipy and SpymMe(2). The minimal inhibitory concentration (MIC) of compounds needed to kill 90% of mycobacterial cells and the IC50 values for the antitumor activity were determined. Compounds 1-4 exhibited good in vitro activity against M. tuberculosis, with MIC values ranging between 0.78 and 6.25 mu g/mL, compared to the free ligands (MIC of 25 to >50 mu g/mL) and the drugs used to treat tuberculosis. Complexes I and 2 also showed promising antitumor activity, with IC50 values of 0.46 +/- 0.02 and 0.43 +/- 0.08 mu M, respectively, against MDA-MB-231 breast tumor cells. (C) 2008 Elsevier Inc. All rights reserved.

A new nitrosyl ruthenium complex: Synthesis, chemical characterization, in vitro and in vivo antitumor activities and probable mechanism of action

This study describes the synthesis of a new ruthenium nitrosyl complex with the formula [RuCl(2)NO(BPA)] [BPA = (2-hydroxybenzyl)(2-methylpyridyl)amine ion], which was synthesized and characterized by spectroscopy, cyclic voltammetry, X-ray crystallography, and theoretical calculation data. The biological studies of this complex included in vitro cytotoxic assays, which revealed its activity against two different tumor cell lines (HeLa and Tm5), with efficacy comparable to that of cisplatin, a metal-based drug that is administered in clinical treatment. The in vivo studies showed that [RuCl2NO(BPA)] is effective in reducing tumor mass. Also, our results suggest that the mechanism of action of [RuCl(2)NO(BPA)] includes binding to DNA, causing fragmentation of this biological molecule, which leads to apoptosis. (C) 2011 Elsevier Masson SAS. All rights reserved.

In vitro Trypanocidal Activity of Phenolic Derivatives from Peperomia obtusifolia

The trypanocidal activity of crude extracts and fractions from the leaves and stems of Peperomia obtusifolia (Piperaceae) was evaluated in vitro against the epimastigote forms of Trypanosoma cruzi. Bioactivity-guided fractionation of the most active extracts afforded seven known compounds, including three chromanes, two furofuran lignans and two flavone C-diglycosides. The most active compounds were the chromanes peperobtusin A and 3,4-dihydro-5-hydroxy-2,7-dimethyl-8-(2 ``-methyl-2 ``-butenyl)-2-(4`-methyl-1`,3`-pentadienyl)-2H-1-benzopyran-6-carboxylic acid, with IC(50) values of 3.1 mu M (almost three times more active than the positive control benznidazole, IC(50) 10.4 mu M) and 27.0 mu M, respectively. Cytotoxicity assays using peritoneal murine macrophages indicated that the chromanes were not toxic at the level of the IC(50) for trypanocidal activity. This is the first report on the trypanocidal activity besides unspecific cytotoxicity of chromanes from Peperomia species. Additionally it represents the first time isolation of 3,4-dihydro5-hydroxy-2,7-dimethyl-8-(2 ``-methyl-2 ``-butenyl)-2-(4`-methyl-1`,3`-pentadienyl)-2H-1-benzopyran-6-carboxylic acid from P. obtusifolia.

Effect of propolis gel on the in vitro reduction of dentin permeability

OBJECTIVE: The aim of this study was to evaluate the capacity of potassium oxalate, fluoride gel and two kinds of propolis gel to reduce the hydraulic conductance of dentin, in vitro. MATERIAL AND METHODS: The methodology used for the measurement of hydraulic conductance of dentin in the present study was based on a model proposed in literature. Thirty-six 1-mm-thick dentin discs, obtained from extracted human third molars were divided into 4 groups (n=9). The groups corresponded to the following experimental materials: GI-10% propolis gel, pH 4.1; GII-30% propolis gel; GIII-3% potassium oxalate gel, pH 4,1; and GIV-1.23% fluoride gel, pH 4.1, applied to the dentin under the following surface conditions: after 37% phosphoric acid and before 6% citric acid application. The occluding capacity of the dentin tubules was evaluated using scanning electron microscopy (SEM) at ×500, ×1,000 and ×2,000 magnifications. Data were analyzed statistically by two-way ANOVA and Tukey's test at 5% significance level. RESULTS: Groups I, II, III, IV did not differ significantly from the others in any conditions by reducing in hydraulic conductance. The active agents reduced dentin permeability; however they produced the smallest reduction in hydraulic conductance when compared to the presence of smear layer (P<0.05). The effectiveness in reducing dentin permeability did not differ significantly from 10% or 30% propolis gels. SEM micrographs revealed that dentin tubules were partially occluded after treatment with propolis. CONCLUSIONS: Under the conditions of this study, the application of 10% and 30% propolis gels did not seem to reduce the hydraulic conductance of dentin in vitro, but it showed capacity of partially obliterating the dentin tubules. Propolis is used in the treatment of different oral problems without causing significant great collateral effects, and can be a good option in the treatment of patients with dentin sensitivity.

In vitro evaluation of an alternative method to bond molar tubes

Despite the advances in bonding materials, many clinicians today still prefer to place bands on molar teeth. Molar bonding procedures need improvement to be widely accepted clinically. OBJECTIVE: The purpose of this study was to evaluate the shear bond strength when an additional adhesive layer was applied on the occlusal tooth/tube interface to provide reinforcement to molar tubes. MATERIAL AND METHODS: Sixty third molars were selected and allocated to the 3 groups: group 1 received a conventional direct bond followed by the application of an additional layer of adhesive on the occlusal tooth/tube interface, group 2 received a conventional direct bond, and group 3 received a conventional direct bond and an additional cure time of 10 s. The specimens were debonded in a universal testing machine. The results were analyzed statistically by ANOVA and Tukey's test (α=0.05). RESULTS: Group 1 had a significantly higher (p<0.05) shear bond strength compared to groups 2 and 3. No difference was detected between groups 2 and 3 (p>0.05). CONCLUSIONS: The present in vitro findings indicate that the application of an additional layer of adhesive on the tooth/tube interface increased the shear bond strength of the bonded molar tubes.

Estudo in vitro da resistência ao cisalhamento da colagem direta de tubos ortodônticos em molares

INTRODUÇÃO: apesar da colagem direta despender menor tempo clínico, com maior preservação da integridade gengival, ainda hoje se observa uma alta incidência de bandagem dos molares. Portanto, torna-se interessante a idealização de recursos para o aumento da eficiência desse procedimento para dentes submetidos a maiores impactos mastigatórios, como, por exemplo, os molares. OBJETIVO: esse estudo teve o propósito de avaliar se a resistência à adesão com a aplicação de uma camada de resina adicional na região oclusal da interface tubo/dente aumenta a qualidade do procedimento de colagem direta de tubos em molares. MÉTODOS: selecionou-se uma amostra composta por 40 terceiros molares inferiores, que foram aleatoriamente divididos em 2 grupos: Grupo 1 - colagem direta convencional, seguida pela aplicação de uma camada de resina na oclusal da interface tubo/dente; e Grupo 2 - colagem direta convencional. O teste de resistência ao cisalhamento foi realizado 24 horas após a colagem, utilizando-se uma máquina de ensaio universal, operando a uma velocidade de 0,5mm/min. Os resultados foram analisados por meio do teste t independente. RESULTADOS: os valores médios obtidos nos testes de cisalhamento foram: 17,08MPa para o Grupo 1 e 12,60MPa para o Grupo 2. O Grupo 1 apresentou uma resistência ao cisalhamento estatisticamente significativa mais alta do que o Grupo 2. CONCLUSÃO: a aplicação de uma camada adicional de resina na oclusal da interface tubo/dente aumenta a qualidade da adesão do procedimento de colagem direta de tubos ortodônticos em molares.

In vitro wear resistance of three types of polymethyl methacrylate denture teeth

The wear resistance of denture teeth is important to the longevity of removable prostheses of edentulous patients. The ability of denture teeth to maintain a stable occlusal relationship over time may be influenced by this property. The purpose of this in vitro study was to evaluate the wear resistance of polymethyl methacrylate (PMMA) denture teeth based on their chemical composition when opposed by a ceramic antagonist. The maxillary canines (n=10) of 3 PMMA denture teeth (Trubyte Biotone, cross-linked PMMA; Trilux, highly cross-linked IPN (interpenetrating polymer network)-PMMA; and Vivodent, highly cross-linked PMMA) were secured in an in vitro 2-body wear-testing apparatus that produced sliding contact of the specimens (4.5 cycles/s, sliding distance of 20 mm, under 37°C running water) against glazed or airborne particle abraded ceramic. Wear resistance was measured as height loss (mm) under 300 g (sliding force) after 100,000 cycles, using a digital measuring microscope. Mean values were analyzed by 2-way ANOVA and Tukey's test (a=0.05). The wear of Trubyte Biotone (0.93 ± 0.14 mm) was significantly higher than that of both other types of teeth tested against abraded ceramic (p<0.05). The Vivodent tooth (0.64 ± 0.17 mm) exhibited the best wear resistance among the denture teeth tested against airborne particle abraded ceramic. There were no statistically significant differences (p>0.05) in wear among the 3 denture teeth evaluated against glazed ceramic. Trilux and Vivodent teeth tested against either glazed or airborne particle abraded ceramic did not differ significantly from each other (p<0.05). All teeth showed significantly more wear against airborne particle abraded ceramic than against glazed ceramic (p<0.05). In conclusion, the three types of PMMA denture teeth presented significantly different wear resistance against the abraded ceramic. The high-strength PMMA denture teeth were more wear-resistant than the conventional PMMA denture tooth.