Cytotoxicity and biocompatibility of a new bioceramic endodontic sealer containing calcium hydroxide

Abstract: This study evaluated the cytotoxicity and biocompatibility of a new bioceramic endodontic sealer (i.e., Sealer Plus BC) in comparison with those of MTA Fillapex and AH Plus. L929 fibroblasts were cultured and Alamar Blue was used to evaluate cell viability of diluted extracts (1:50, 1:100, and 1:200) from each sealer at 24 h. Polyethylene tubes that were filled with material or empty (as a control) were implanted in the subcutaneous tissue of rats. The rats were killed after 7 and 30 d (n = 8), and the tubes were removed for histological analysis. Parametric data was analyzed using a one-way ANOVA test, and nonparametric data was analyzed via the Kruskal-Wallis test followed by the Dunn test (p < 0.05). A reduction in cell viability was observed in the extracts that were more diluted for Sealer Plus BC when compared to that of Control and AH Plus (p < 0.05). However, the 1:50 dilution of the Sealer Plus BC was similar to that of the Control (p > 0.05). Conversely, more diluted extracts of MTA Fillapex (1:200) and AH Plus (1:100 and 1:200) were similar to the Control (p > 0.05). Histological analysis performed at 7 d did not indicate any significant difference between tissue response for all materials, and the fibrous capsule was thick (p > 0.05). At 30 d, Sealer Plus BC was similar to the Control (p > 0.05) and MTA Fillapex and AH Plus exhibited greater inflammation than the Control (p < 0.05). The fibrous capsule was thin for the Control and for most specimens of Sealer Plus BC and AH Plus. Thus, Sealer Plus BC is biocompatible when compared to MTA Fillapex and AH Plus, and it is less cytotoxic when less-diluted extracts are used.

The deubiquitinase USP38 affects cellular functions through interacting with LSD1

BACKGROUND: Deubiquitination is a posttranslational protein modification prevalent in mammalian cells. Deubiquitinases regulate the functions of the target protein by removing its ubiquitin chain. In this study, the effects of the deubiquitinase USP38's functions on the LSD1 protein and on cell physiology were investigated. MATERIALS AND METHODS: Western blotting, real-time quantitative PCR, immunoprecipitation, denaturing immunoprecipitation and luciferase reporter assays were used to analyze the protein stability, protein interactions and changes in the ubiquitin chain. Cell proliferation assays, colony formation assays, drug treatments and western blotting were used to explore the functions of USP38 in cells. RESULTS: The deubiquitinase USP38 stabilizes protein LSD1 in cells by binding LSD1 and cleaving its ubiquitin chain to prevent the degradation of LSD1 by the intracellular proteasome. USP38 enhances the ability of LSD1 to activate signaling pathways and hence promotes cellular abilities of proliferation and colony formation through interacting with LSD1. Furthermore, USP38 enhances the drug tolerance of human colon cancer cells. CONCLUSIONS: USP38 is an LSD1-specific deubiquitinase that affects cellular physiology through interacting with LSD1.

Physical properties and biological effects of mineral trioxide aggregate mixed with methylcellulose and calcium chloride

Abstract Objectives: Methylcellulose (MC) is a chemical compound derived from cellulose. MTA mixed with MC reduces setting time and increases plasticity. This study assessed the influence of MC as an anti-washout ingredient and CaCl2 as a setting time accelerator on the physical and biological properties of MTA. Material and Methods: Test materials were divided into 3 groups; Group 1(control): distilled water; Group 2: 1% MC/CaCl2; Group 3: 2% MC/CaCl2. Compressive strength, pH, flowability and cell viability were tested. The gene expression of bone sialoprotein (BSP) was detected by RT-PCR and real­ time PCR. The expression of alkaline phosphatase (ALP) and mineralization behavior were evaluated using an ALP staining and an alizarin red staining. Results: Compressive strength, pH, and cell viability of MTA mixed with MC/CaCl2 were not significantly different compared to the control group. The flowability of MTA with MC/CaCI2 has decreased significantly when compared to the control (p<.05). The mRNA level of BSP has increased significantly in MTA with MC/CaCl2 compared to the control (p<.05). This study revealed higher expression of ALP and mineralization in cells exposed to MTA mixed with water and MTA mixed with MC/CaCl2 compared to the control (p<.05). Conclusions: MC decreased the flowability of MTA and did not interrupt the physical and biological effect of MTA. It suggests that these cements may be useful as a root-end filling material.

Characterization of cryopreserved primary human corneal endothelial cells cultured in human serum-supplemented media / Caracterização de células endoteliais corneanas humanas primárias criopreservadas cultivadas em meio suplementado com soro humano

ABSTRACT Purpose: To compare cryopreserved human corneal endothelial cells (HCECs) grown in human serum-supplemented media (HS-SM) with cryopreserved HCECs grown in fetal bovine serum-supplemented media (FBS-SM). Methods: Three pairs of human corneas from donors aged 8, 28, and 31 years were obtained from the eye bank. From each pair, one cornea was used to start a HCEC culture using HS-SM; the other cornea was grown in FBS-SM. On reaching confluence, the six cell populations were frozen using 10% dimethyl sulfoxidecontaining medium. Thawed cells grown in HS-SM were compared with those grown in FBS-SM with respect to morphology, growth curves, immunohistochemistry, real time-reverse transcriptase polymerase chain reaction (RT-PCR) for endothelial cell markers, and detachment time. Results: No difference in morphology was observed for cells grown in the two media before or after cryopreservation. By growth curves, cell counts after thawing were similar in both media, with a slight trend toward higher cell counts in FBS-SM. Cells grown in both the media demonstrated a similar expression of endothelial cell markers when assessed by immunohistochemistry, although HCEC marker gene expression was higher in cells grown in HS-SM than in those grown in FBS-SM as assessed by RT-PCR. With FBS-SM, there was a tendency of longer detachment time and lower cell passages. Conclusions: HS-SM was similar to FBS-SM for cryopreservation of cultured HCECs as assessed by analysis of cell morphology, proliferation, and protein expression, although marker gene expression was higher in cells grown in HS-SM than in those grown in FBS-SM. Detachment time was longer with FBS-SM and in lower passages.

RESUMO Objetivo: Comparar células endoteliais de córnea humana (HCECs) criopreservadas e cultivadas em meio suplementado com soro humano (HS-SM) com HCEC criopreservadas e cultivadas em meio suplementado com soro bovino fetal (FBS-SM). Métodos: Três pares de córneas humanas de doadores com 8, 28 e 31 anos de idade foram obtidos do banco de olhos e, de cada par, uma córnea foi utilizado para iniciar uma cultura com HS-SM e outra com FBS-SM. Ao atingir a confluência, as populações de células foram congeladas utilizando-se dimetil-sulfóxido 10% no respectivo meio de cultura. Após descongeladas, as células cultivadas em HS-SM foram comparados com as cultivadas em FBS-SM por meio de morfologia, curva de crescimento, imuno-histoquímica, reação em cadeia de Reação em cadeia da polimerase da transcrição reversa em tempo real (RT-PCR) para marcadores de células endoteliais e tempo de descolamento. Resultado: Não foram observadas diferenças morfológicas antes ou após a criopreservação. Curva de crescimento mostrou contagens celulares semelhantes em ambos os meios, com discreta tendência para um maior número em FBS-SM. As células cultivadas em ambos os meios mostraram expressão semelhante de marcadores celulares endoteliais quando avaliadas por imuno-histoquímica, embora a expressão genética de marcadores para HCEC tenha sido maior em HS-SM quando avaliado por RT-PCR. Houve uma tendência de maior tempo de descolamento com FBS-SM e passagens iniciais. Conclusões: HS-SM foi semelhante ao FBS-SM na criopreservação de HCEC cultivadas in vitro quando avaliadas por morfologia celular, proliferação celular e expressão proteica, embora a expressão genética de marcadores endoteliais tenha sido maior em células cultivadas em HS-SM quando comparadas a células cultivadas em FBS-SM. O tempo de descolamento foi maior quando utilizado FBS-SM e em passagens iniciais.

Cytotoxic effects of new MTA-based cement formulations on fibroblast-like MDPL-20 cells

Abstract The present study aimed at evaluating the cytotoxic effects of a novel cement called CER on periodontal fibroblast-like cells of mice (MDPL-20), in comparison with different formulations of Mineral Trioxide Aggregate (MTA), by means of the cell viability test (MTT) and cell morphology analysis. Thirty-two round-shaped samples were fabricated with the following cements: white MTA, white and gray CER and experimental white MTA. The samples were immersed in serum-free culture medium for 24 hours or 7 days (n = 16). The extracts (culture medium + components released from the cements) were applied for 24 hours to previously cultured cells (40.000 cells/cm2) in the wells of 24-well plates. Cells seeded in complete culture medium were used as a negative control. Cell viability was assessed using the MTT assay. Two samples of each cement were used for cell morphology analysis by Scanning Electron Microscopy (SEM). The extracts obtained at the 7-day period presented higher cytotoxicity compared with the 24-hour period (p < 0.05). The gray CER obtained at 24 hours presented the highest cytotoxic effect, whereas the experimental white MTA presented the lowest, similar to the control (p > 0.05). However, at the 7-day period, the experimental white MTA presented no significant difference in comparison with the other cements (p > 0.05). At the 7-day period, CER cement presented cytotoxic effects on fibroblast-like cells, similar to different MTA formulations. However, the immersion period in the culture medium influenced the cytotoxicity of the cements, which was greater for CER cement at 24 hours.

Effects of fluoride on proliferation and mineralization in periodontal ligament cells in vitro

Fluoride, which is often added to toothpaste or mouthwash in order to protect teeth from decay, may be a novel therapeutic approach for acceleration of periodontal regeneration. Therefore, we investigated the effects of fluoride on proliferation and mineralization in human periodontal ligament cells in vitro. The periodontal ligament cells were stimulated with various concentrations of NaF added into osteogenic inductive medium. Immunohistochemistry of cell identification, cell proliferation, alkaline phosphatase (ALP) activity assay, Alizarin red S staining and quantitative real-time-polymerase chain reaction (RT-PCR) were performed. Moderate concentrations of NaF (50-500 μmol/L) had pro-proliferation effects, while 500 μmol/L had the best effects. ALP activity and calcium content were significantly enhanced by 10 μmol/L NaF with osteogenic inductive medium. Quantitative RT-PCR data varied in genes as a result of different NaF concentrations and treatment periods. We conclude that moderate concentrations of NaF can stimulate proliferation and mineralization in periodontal ligament cells. These in vitro findings may provide a novel therapeutic approach for acceleration of periodontal regeneration by addition of suitable concentrations of NaF into the medication for periodontitis treatment, i.e., into periodontal packs and tissue patches.

Biological and mechanical properties of an experimental glass-ionomer cement modified by partial replacement of CaO with MgO or ZnO

AbstractSome weaknesses of conventional glass ionomer cement (GIC) as dental materials, for instance the lack of bioactive potential and poor mechanical properties, remain unsolved.Objective The purpose of this study was to investigate the effects of the partial replacement of CaO with MgO or ZnO on the mechanical and biological properties of the experimental glass ionomer cements.Material and Methods Calcium fluoro-alumino-silicate glass was prepared for an experimental glass ionomer cement by melt quenching technique. The glass composition was modified by partial replacement (10 mol%) of CaO with MgO or ZnO. Net setting time, compressive and flexural properties, and in vitrorat dental pulp stem cells (rDPSCs) viability were examined for the prepared GICs and compared to a commercial GIC.Results The experimental GICs set more slowly than the commercial product, but their extended setting times are still within the maximum limit (8 min) specified in ISO 9917-1. Compressive strength of the experimental GIC was not increased by the partial substitution of CaO with either MgO or ZnO, but was comparable to the commercial control. For flexural properties, although there was no significance between the base and the modified glass, all prepared GICs marked a statistically higher flexural strength (p<0.05) and comparable modulus to control. The modified cements showed increased cell viability for rDPSCs.Conclusions The experimental GICs modified with MgO or ZnO can be considered bioactive dental materials.

Neuropeptide Y, substance P, and human bone morphogenetic protein 2 stimulate human osteoblast osteogenic activity by enhancing gap junction intercellular communication

Bone homeostasis seems to be controlled by delicate and subtle “cross talk” between the nervous system and “osteo-neuromediators” that control bone remodeling. The purpose of this study was to evaluate the effect of interactions between neuropeptides and human bone morphogenetic protein 2 (hBMP2) on human osteoblasts. We also investigated the effects of neuropeptides and hBMP2 on gap junction intercellular communication (GJIC). Osteoblasts were treated with neuropeptide Y (NPY), substance P (SP), or hBMP2 at three concentrations. At various intervals after treatment, cell viability was measured by the MTT assay. In addition, cellular alkaline phosphatase (ALP) activity and osteocalcin were determined by colorimetric assay and radioimmunoassay, respectively. The effects of NPY, SP and hBMP on GJIC were determined by laser scanning confocal microscopy. The viability of cells treated with neuropeptides and hBMP2 increased significantly in a time-dependent manner, but was inversely associated with the concentration of the treatments. ALP activity and osteocalcin were both reduced in osteoblasts exposed to the combination of neuropeptides and hBMP2. The GJIC of osteoblasts was significantly increased by the neuropeptides and hBMP2. These results suggest that osteoblast activity is increased by neuropeptides and hBMP2 through increased GJIC. Identification of the GJIC-mediated signal transduction capable of modulating the cellular activities of bone cells represents a novel approach to studying the biology of skeletal innervation.

Synthesis and cytotoxicity evaluation of granular magnesium substituted beta-tricalcium phosphate

Objective: The aim of this study was to produce dense granules of tricalcium phosphate (β-TCP) and magnesium (Mg) substituted β-TCP, also known as β-TCMP (Mg/Ca=0.15 mol), in order to evaluate the impact of Mg incorporation on the physicochemical parameters and in vitro biocompatibility of this novel material. Material and Methods: The materials were characterized using X-ray diffraction (XRD), infrared spectroscopy (FTIR), electron microscopy and inductively coupled plasma (ICP). Biocompatibility was assayed according to ISO 10993-12:2007 and 7405:2008, by two different tests of cell survival and integrity (XTT and CVDE). Results: The XRD profile presented the main peaks of β-TCP (JCPDS 090169) and β-TCMP (JCPDS 130404). The characteristic absorption bands of TCP were also identified by FTIR. The ICP results of β-TCMP granules extract showed a precipitation of calcium and release of Mg into the culture medium. Regarding the cytotoxicity assays, β-TCMP dense granules did not significantly affect the mitochondrial activity and relative cell density in relation to β-TCP dense granules, despite the release of Mg from granules into the cell culture medium. Conclusion: β-TCMP granules were successfully produced and were able to release Mg into media without cytotoxicity, indicating the suitability of this promising material for further biological studies on its adequacy for bone therapy.

Mayor frecuencia de aberraciones cromosómicas en linfocitos expuestos o no a mitomicina C, de mujeres posmenopáusicas obesas en comparación con mujeres no obesas del departamento del Cauca, Colombia / High frequency of chromosome aberrations observed in lymphocytes in postmenopausal obese women

Introducción. Los estudios epidemiológicos indican que la obesidad está asociada en el 25 al 30 % con varios tipos de cáncer. Objetivo. Evaluar la frecuencia de aberraciones cromosómicas en linfocitos de mujeres posmenopáusicas obesas y no obesas, mediante la prueba de reto celular (challenge assay) como biomarcador de inestabilidad genómica. Materiales y métodos. Cuarenta mujeres posmenopáusicas fueron incluidas en el estudio (20 obesas y 20 no obesas). Los grupos fueron pareados según edad (± 5 años) y procedencia. Después de la firma voluntaria del consentimiento informado, las mujeres fueron entrevistadas y se les tomó una muestra de 5 ml de sangre periférica. Se establecieron cultivos de linfocitos con tratamiento con mitomicina C y sin él (prueba de reto celular) y, posteriormente, se registró la frecuencia de aberraciones cromosómicas para cada grupo y tratamiento. Resultados. En general, las mujeres obesas presentaron una mayor frecuencia de aberraciones cromosómicas en comparación con las no obesas. Después de exponer los cultivos celulares a mitomicina C, las mujeres obesas presentaron un incremento en el número de aberraciones cromosómicas totales en comparación con las no obesas (3,74±0,63 Vs. 2,70±0,61; p=0,001). Conclusiones. La mayor frecuencia de aberraciones cromosómicas en los linfocitos de mujeres posmenopáusicas obesas que en no obesas, sugiere diferencias en la capacidad de reparación del ADN, lo cual podría explicar la asociación entre la inestabilidad genómica y la mayor incidencia de cáncer en esta población.

Introduction. Epidemiological studies indicate that obesity is associated with an increased risk of 20-25% with several types of cancer. Objective. The frequency of chromosome aberrations was evaluated in lymphocytes from postmenopausal obese and non-obese women. Materials and methods. Twenty obese and 20 non-obese women, all post-menopause, were recruited. The groups were matched according to age (± 5 years) and place of origin. After signing the consent form, women were interviewed using a structured questionnaire, and a blood sample (5 ml) was drawn into vacutainer tubes. From each sample, lymphocyte cell cultures were established with and without mitomycin C (challenge assay). Afterwards, the frequency of chromosome aberrations were recorded for each group and treatment. Data were analyzed using the statistical program SPSS, v. 14.0. Results. Obese women had a higher frequency of chromosome aberrations when compared with non-obese women. After exposing the cell cultures to mitomycin C, obese women presented an increase in the number of total chromosome aberrations in comparison to non-obese women (3.7± 0.6 vs. 2.70±0.6; p=0.001). Conclusions. The higher frequency of chromosome aberrations in lymphocytes from postmenopausal obese women compared to non-obese women suggested differences in the DNA repair capacity. This may indicate an association between genomic instability and the higher incidence of cancer in this population.

Interacciones genotóxicas de mutágenos en mezclas binarias mediante ensayo cometa alcalino en linfocitos humanos / Interaction of mutagens in binary mixtures using the alkaline comet assay in human lymphocytes

Introducción. Los mutágenos contenidos en mezclas complejas presentan interacciones de sinergismo, aditivas o antagónicas. Se han desarrollado enfoques experimentales que permitan dilucidar el responsable de las interacciones en la mezcla. Objetivo. Desarrollar un diseño experimental para comprender los procesos que se llevan a cabo entre los compuestos presentes en las mezclas complejas. Materiales y métodos. Se expusieron linfocitos humanos a mezclas binarias de mutágenos B[a]P, DMBA, Trp-P-1 y MX durante una hora, con activación metabólica y sin ella. La viabilidad se evaluó con azul de tripano y, la genotoxicidad, con cometa alcalino. Resultados. Ningún hidrocarburo tuvo efecto con furanona. Con S9 y sin él, se observó que se presentaban interacciones tóxicas entre hidrocarburos. Se observó sinergismo sin S9 entre B[a]P y Trp-P-1 y, con actividad metabólica, entre DMBA y Trp-P-1. Sin S9 se observó interacción antagónica entre Trp-P-1 y DMBA y, con S9, entre Trp-P-1 y MX y entre MX y DMBA. Se observó un incremento dependiente de la dosis en la longitud de la cola. Hubo daño genotóxico medio y aumento de las células dañadas. Para todas las mezclas se pudo determinar la concentración mínima en la que se observaban efectos adversos y solo para algunas se determinó la concentración máxima en la cual no se observaron efectos adversos. Conclusión. Se hace un aporte para comprender los procesos que ocurren cuando en una mezcla hay presentes, al menos, dos mutágenos y se valida un modelo de análisis que permite dilucidar el compuesto que tiene efecto sobre otro. También, se demostró que según el tipo de compuestos en la mezcla, se tendrá o no un umbral de riesgo.

Introduction. Mutagens contained in complex mixtures can present synergistic interactions, either additive or antagonistic. Therefore, development of experimental approaches is necessary to elucidate which is the responsible agent for the effect in the mixtures. Objective. An experimental design was developed that allowed an understanding of the processes between the compounds of complex mixtures. Materials and methods. Human lymphocytes were exposed to binary mixtures of the mutagens B[a]P, DMBA, Trp-P-1 and MX for 1 hour with or without S9. Viability was assessed with trypan blue dye and the genotoxicity by the comet assay. Results. All of the hydrocarbon showed an effect with furanone. With and without S9, the most toxic interactions were observed between hydrocarbons. Synergistic interaction was observed without S9 between B [a] P and Trp-P-1 and between DMBA and Trp-P-1 with metabolic activity. Without S9 antagonistic interaction was observed only between Trp-P-1+DMBA, and with S9 between Trp-P-1+MX and MX+DMBA. It observed an increase dose dependent in tail length. Half the cultures showed genotoxic damage and increased cell damage. For each mixture, minimum concentrations were determined at which adverse effects are observed; for some only the maximum concentration was determined at which no adverse effects are observed. Conclusion. The processes between mutagens present in a mixture have become better understood, and the results validated an analytical model that determined which component had an effect on another. The results also showed that the type of compounds in the mixture determined whether or not a risk threshold was present.

A simple method to measure cell viability in proliferation and cytotoxicity assays

Resazurin dye has been broadly used as indicator of cell viability in several types of assays for evaluation of the biocompatibility of medical and dental materials. Mitochondrial enzymes, as carriers of diaphorase activities, are probably responsible for the transference of electrons from NADPH + H+ to resazurin, which is reduced to resorufin. The level of reduction can be quantified by spectrophotometers since resazurin exhibits an absorption peak at 600 çm and resorufin at 570 çm wavelengths. However, the requirement of a spectrophotometer and specific filters for the quantification could be a barrier to many laboratories. Digital cameras containing red, green and blue filters, which allow the capture of red (600 to 700 çm) and green (500 to 600 çm) light wavelengths in ranges bordering on resazurin and resorufin absorption bands, could be used as an alternative method for the assessment of resazurin and resorufin concentrations. Thus, our aim was to develop a simple, cheap and precise method based on a digital CCD camera to measure the reduction of resazurin. We compared the capability of the CCD-based method to distinguish different concentrations of L929 and normal Human buccal fibroblast cell lines with that of a conventional microplate reader. The correlation was analyzed through the Pearson coefficient. The results showed a strong association between the measurements of the method developed here and those made with the microplate reader (r² = 0.996; p < 0.01) and with the cellular concentrations (r² = 0.965; p < 0.01). We concluded that the developed Colorimetric Quantification System based on CCD Images allowed rapid assessment of the cultured cell concentrations with simple equipment at a reduced cost.

In vitro anti-microbial activity of the Cuban medicinal plants Simarouba glauca DC, Melaleuca leucadendron L and Artemisia absinthium L

In the present study, an extensive in vitro antimicrobial profiling was performed for three medicinal plants grown in Cuba, namely Simarouba glauca, Melaleuca leucadendron and Artemisia absinthium. Ethanol extracts were tested for their antiprotozoal potential against Trypanosoma b. brucei, Trypanosoma cruzi, Leishmania infantum and Plasmodium falciparum. Antifungal activities were evaluated against Microsporum canis and Candida albicans whereas Escherichia coli and Staphylococcus aureus were used as test organisms for antibacterial activity. Cytotoxicity was assessed against human MRC-5 cells. Only M. leucadendron extract showed selective activity against microorganisms tested. Although S. glauca exhibited strong activity against all protozoa, it must be considered non-specific. The value of integrated evaluation of extracts with particular reference to selectivity is discussed.

Cytotoxic effect of a new endodontic cement and mineral trioxide aggregate on L929 line culture

The aim of this study was to compare the cytotoxicity of Mineral Trioxide Aggregate [MTA] and a New Endodontic Cement [NEC] on L929 mouse fibroblasts. Different dilutions [Neat, 1/2, 1/10, 1/100] of fresh and set materials placed adjacent flasks of L929 in DMEM medium. Cellular viability was assessed using MTT assay in three time intervals [24, 48, and 72 h after mixing]. Differences in mean cell viability values between materials were assessed by using the One-way ANOVA and Bonferoni post-test. Optical microscopic analysis of morphology of the untreated control and the cementtreated cell cultures were carried out in all experimental periods. It was indicated that there was not a significant difference in cytotoxicity among the materials of test and between them and the control group. However, there was a statistically significant difference between different time intervals within each group [P

Evaluation of genotoxic potential of chromium (VI) in Channa punctata fish in terms of chromosomal aberrations.

Chromium, a widely recognized carcinogenic, mutagenic and redox active metal, is released into aquatic environments by electroplating, tannery and textile industries. Elevated concentrations in sediments and interstitial waters are well documented. Fishes dwelling in chromium waste infested waters are presumed to be affected by its deposits. To evaluate the genotoxic potential of chromium [Cr(VI)] on aquatic bio-system, bottom feeding fishes, Channa punctata, as model fish, were exposed to [Cr(VI)]. The chromosomal aberration test (CAT) was used as biomarker of [Cr(VI)] induced toxicity. The fish were divided into three groups:Group I non-treated controls; group II positive controls, treated with an intra-muscular injection of mitomycin-C at 1 mg/kg body wt; group III exposed to a sublethal concentration (7.689 mg/l) of [Cr(VI)], dissolved in the water. For CAT estimation, short term static bioassays were conducted and samples were collected from the kidneys of fish after 24, 48, 72, 96 and 168 hrs of exposure. The remarkable chromosomal aberrations recorded in the present investigation included chromatid breaks, chromosome breaks, chromatid deletions, fragments, acentric fragments, and ring and di-centric chromosomes, along with chromatid and chromosome gaps. A significant increase in chromosomal aberrations was observed after 72 hrs of [Cr(VI)] exposure. The present study, thus reveals that even for acute exposure, [Cr(VI)] is a genotoxic agent for C. punctata.

In vitro biocompatibility tests of two commercial types of mineral trioxide aggregate / Testes de biocompatibilidade in vitro de duas formas comerciais do agregado de trióxido mineral

Recentemente, o agregado de trióxido mineral (MTA) regular e branco estão sendo utilizados na Odontologia como materiais para obturação retrógrada de canais radiculares. Testes de genotoxicidade e citotoxicidade formam uma importante parte da pesquisa do câncer e da avaliação de risco de carcinógenos potenciais. Assim, o objetivo deste estudo foi examinar a genotoxicidade e citotoxicidade do MTA branco e regular in vitro pelo teste do cometa e teste de exclusão pelo azul de tripan, respectivamente. Células do linfoma murino foram expostas às duas formas de apresentação do MTA nas concentrações finais de 1 a 1.000 µg/mL por 3 horas a 37ºC. Os resultados mostraram que ambos os compostos testados não produziram efeito genotóxico em todas as concentrações testadas. Da mesma forma, nenhuma diferença estatisticamente significativa (p > 0,05) foi observada na citotoxicidade. Em suma, nossos resultados sugerem que o MTA regular e branco não são genotoxinas e não são capazes de interferir na viabilidade celular conforme avaliado pelo teste do cometa e ensaio do azul de tripan, respectivamente.

Theiler virus-GDVII strain (TMEV-GDVII) infection of cultured astrocytes. An image analysis of its effects on cell activation

Our original aim was to determine whether dBcAMP-induced activation of cultured astrocytes affected the course of subsequent viral infection. After 2 h exposure of 2-day-old first subculture of mouse astrocytes to dBcAMP 1 mM, cell monolayers grown in glass coverslips of Leighton tubes were inoculated with 10(3) PFU of Theiler virus-GDVII strain (TMEV-GDVII). At 9 days post-infection (pi), viral infectivity persisted in supernatants from dBcAMP-treated cultures, but was no longer detectable in non-stimulated controls. The relatively spared astroglial monolayer at day 1 pi, hardly affected by progressive viral cytolytic effect, was chosen for immunolabeled cell count, whether by viral antigen or GFAP. To this end, 20 fields for each coverslip were digitalized at 250x final magnification. In dBcAMP treated cultures, viral antigen(+) cells were fewer and lower in percentage versus infected cultures lacking stimulation. As regards GFAP staining, stimulation or infection per se induced a greater number and percentage of labeled astrocytes. According to morphometric characterization, such increase was due to a greater number of process-bearing astrocytes. It may be concluded that, regardless of previous dBcAMP treatment, early TMEV-GDVII infection enhanced immunocytochemical and morphological differentiation in cultured astrocytes.

Preliminary results on embryo rescue for circumventing hybridization barriers in Asparagus

Garden asparagus, Asparagus officinalis, is reproductively isolated from a related ornamental species with potential breeding value, Asparagus densiflorus cv. Sprengeri, by pre- and post-zygotic barriers. The latter barrier operates at the endosperm level five days after pollination in A. officinalis x A. densiflorus crosses. To try to circumvent this barrier, in vitro embryo rescue using ovule and ovary cultures was tested. Controlled interspecific crosses were made and 2,032 ovules and 826 ovaries were cultured three days after pollination under various culture media and incubation conditions. Ovaries cultured for 60 days became red (similar to mature fruits), but seed formation was incomplete. Transfer of ovules to other media was necessary to promote embryo development. The interspecific embryos increased their length from 35 microns at the initiation of culture to 1,900 microns after 120 days of culture, but seedlings were not obtained. Histological studies revealed differentiation of protoderm only. The possible causes of the failure of the embryos to complete differentiation and morphogenesis are discussed.

Morphogenesis in short-term and long-term anther derived calli of Oenothera hookeri de vries

Anther culture of O. hookeri on Murashige and Skoog (1962) medium supplemented with 2 mg l-1 2,4-dichlorophenoxyacetic acid and 2 mg-1 1-naphthaleneacetic acid produced callus formation. When subcultured onto medium lacking auxin, the callus regenerated through the organogenic pathway. Non-organogenic and organogenic callus was observed using histological methods after 2, 3 and 24 weeks in culture. Three types of calli were recognized: non-organogenic friable calli, organogenic friable calli with roots and organogenic hard calli with shoots. The microscopical sections showed striking differences in tissue organization among friable and compact calli. Vascular bundles were prominent in compact calli, but were not found in friable calli. Calli sections showed at light microscopy cells at two developmental stages; differentiated highly vacuolated cells and meristematic small isodiametric cells with densely stained cytoplasm. At electron microscopy level abnormal chloroplasts were present in non-organogenic calli, while chloroplasts were well developed in organogenic hard calli. Peroxisomes with paracrystalline protein bodies were abundant in both types of calli.