DRUG DEVELOPMENT RESEARCH, Issue 4 2008
Anna Galkin
Abstract New apoptosis modulating agents are widely sought, because failure in regulation of apoptosis is associated with many diseases. In this study, we have evaluated apoptosis inducing the potential of ten new hydrophobic derivatives of 5-(hydroxymethyl)isophthalic acid. Cancerous leukemia cells (HL-60) and non-malignant fibroblasts (Swiss 3T3) were incubated with test compounds for 24,h and morphologically evaluated. The changes in mitochondrial membrane potential (,,m) and caspase-3 activity were used to confirm the results and to study early induction of apoptosis. Cytotoxicity was determined using the lactate dehydrogenase (LDH) assay and mutagenicity with miniaturized Ames-test. The most potent selective apoptosis inducers were compounds 1c and 1,h having IC50 values of 41 and 23,µM, respectively, in leukemia cells (HL-60) while effects in fibroblasts (Swiss 3T3) were insignificant. Reduction of ,,m and increase in caspase-3 activity were observed already during the first 2,hr in the HL-60 cells treated with compounds 1,c and 1,h. Neither of the compounds was cytotoxic or mutagenic. The results indicate that compounds 1,c and 1,h are selective apoptosis inducers and should be studied further for possible use in cancer therapy. Drug Dev. Res. 69: 185,195, 2008. © 2008 Wiley-Liss, Inc. [source]

Cytotoxicity of doxorubicin-loaded Con A-liposomes

DRUG DEVELOPMENT RESEARCH, Issue 5 2006
Hercília Maria Lins Rolim Santos
Abstract The present study investigated the potential of Concanavalin A lectin (Con A) conjugated to liposomes (Con A-liposomes) for targeting doxorubicin (DOX) to cells. The physicochemical properties and the cytotoxicity of DOX-loaded Con A-liposomes were evaluated. DOX-loaded Con A-liposomes were prepared by incubation of DOX-loaded liposomes with a Con A-SATA derivative. Lectin biological activity was monitored before and after conjugation by a hemagglutinating assay. The cytotoxicity of DOX-loaded Con A-liposomes was evaluated in terms of the inhibition of NCI-H299 and HEp-2 cell proliferation using the MTT method. The affinity of lectinized liposomes with these cells was thus assessed by evaluating the cytotoxic effect of the DOX released into cells. Stable DOX-loaded Con A-liposomes were obtained and their high affinity for cells was corroborated. The encapsulation of DOX into Con A-liposomes produced an inhibition of roughly 70% of Hep-2 cell proliferation and 50% of cell inhibition was verified on HCI-H292. DOX in solution was able to inhibit only 20% of cell proliferation for both cell lines. Unloaded Con A-liposomes were not cytotoxic. The encapsulation of DOX into Con A-liposomes improves drug penetration into cells, thereby enhancing its cytotoxicity, especially in Hep-2 cells. Drug Dev. Res. 67:430,437, 2006. © 2006 Wiley-Liss, Inc. [source]

Cytotoxicity and oxidative stress caused by chemicals adsorbed on particulate matter,

ENVIRONMENTAL TOXICOLOGY, Issue 5 2006
Andrea Müller
Abstract Air particulate matter (PM) and bound chemicals are potential mediators for adverse health effects. The cytotoxicity and changes in energy-providing processes caused by chemical compounds bound to PM of different size fractions were investigated in Tetrahymena pyriformis. The PM samplings were carried out using a high volume cascade impactor (6 size fractions between 10 ,m and less than 0.49 ,m) at three points of La Plata, Argentina: in an industrial area, a traffic-influenced urban area, and a control area. Extracts from respirable particles below 1 ,m initiated the highest cytotoxic effects, demonstrating their higher risk. In contrast, an increase on oxygen consumption was observed especially in tests of extracts from particles less than 1 ,m from urban and industrial areas. The increase on oxygen consumption could be caused by decoupling processes in the respiratory chain. Otherwise the ATP concentration was increased too, even though to a lower extent. The observed imbalance between oxygen consumption and ATP concentration in exposed T. pyriformis cells may be due to oxidative stress, caused by chemical compounds bound to the particles. Owing to the complexity of effects related to PM and their associated chemical compounds, various physiological parameters necessarily need to be investigated to obtain more information about their possible involvement in human relevant pathogenic processes. As shown here, effects on cell proliferation and on energy-providing processes are suitable indicators for the different impact of PM and adsorbed chemicals from various sampling locations. © 2006 Wiley Periodicals, Inc. Environ Toxicol 21: 457,463, 2006. [source]

Cytotoxicity of settling particulate matter and sediments of the Neckar River (Germany) during a winter flood

ENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 3 2000
Henner Hollert
Abstract To investigate the cytotoxic and genotoxic potentials of settling particulate matter (SPM) carried by the Neckar River, a well-studied model for a lock-regulated river in central Europe, during a flood, acute cytotoxicity was investigated using the fibroblast-like fish cell line RTG-2 with the neutral red retention, the succinic acid dehydrogenase (MTT), and the lactatedehydro-genase (LDH) release assays as well as microscopic inspection as endpoints. Genotoxicity of water, pore water, sediments, and SPM were assessed using the Ames test. Different extraction methods (Soxhlet extraction with solvents of variable polarity as well as a fluid/fluid extraction according to pH) in addition to a supplementation of biotests with S9 fractions from the liver of ,-naphthoflavone/phenobarbital-induced rats allowed a further characterization of the biological damage. Both sediments and SPM extracts caused cytotoxic effects in RTG-2 cells. Cytotoxicity was found to increase significantly with polarity of extracting solvents (NR50 = effective concentration for 50% cell death in the neutral red test: 80 [65], 100 [70], 180 [220], and 225 [270] mg/ml for ethanol, acetone, dichloromethane, and n -hexane extracts, respectively, if measured with [without] S9 supplementation). Following extraction according to pH, cytotoxicity could be attributed mainly to neutral substances (NR50: 80 and 218 mg dry SPM/ml test medium for the neutral and the acid fractions, respectively), whereas the slightly acid and basic fractions already showed little or no cytotoxicity. Samples taken during the period of flood rise showed the highest cytotoxic activities. Cytotoxicity was significantly enhanced by the addition of S9 preparations. In contrast, no genotoxic activity was found in native surface waters, pore waters, and SPM. [source]

Iron enhances endothelial cell activation in response to Cytomegalovirus or Chlamydia pneumoniae infection

EUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 10 2006
A. E. R. Kartikasari
Abstract Background, Chronic inflammation has been implemented in the pathogenesis of inflammatory diseases like atherosclerosis. Several pathogens like Chlamydia pneumoniae (Cp) and cytomegalovirus (CMV) result in inflammation and thereby are potentially artherogenic. Those infections could trigger endothelial activation, the starting point of the atherogenic inflammatory cascade. Considering the role of iron in a wide range of infection processes, the presence of iron may complicate infection-mediated endothelial activation. Materials and methods, Endothelial intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and endothelial selectin (E-selectin) expression were measured using flow cytometry, as an indication of endothelial activation. Cytotoxicity was monitored using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Immunostaining was applied to measure Cp and CMV infectivity to endothelial cells. Results, An increased number of infected endothelial cells in a monolayer population leads to a raised expression of adhesion molecules of the whole cell population, suggesting paracrine interactions. Iron additively up-regulated Cp-induced VCAM-1 expression, whereas synergistically potentiated Cp-induced ICAM-1 expression. Together with CMV, iron also enhanced ICAM-1 and VCAM-1 expression. These iron effects were observed without modulation of the initial infectivity of both microorganisms. Moreover, the effects of iron could be reversed by intracellular iron chelation or radical scavenging, conforming modulating effects of iron on endothelial activation after infections. Conclusions, Endothelial response towards chronic infections depends on intracellular iron levels. Iron status in populations positive for Cp or CMV infections should be considered as a potential determinant for the development of atherosclerosis. [source]

Synthesis, Cytotoxicity and Antibacterial Studies of p -Methoxybenzyl-Substituted and Benzyl-Substituted N-Heterocyclic Carbene,Silver Complexes

EUROPEAN JOURNAL OF INORGANIC CHEMISTRY, Issue 7 2010
Siddappa Patil
Abstract p -Methoxybenzyl-substituted and benzyl-substituted N-heterocyclic carbene (NHC) [(3a,c) and (6a,c)] precursors were synthesised from the reaction of 1H -imidazole (1a), 4,5-dichloro-1H -imidazole (1b), and 1H -benzimidazole (1c) with p -methoxybenzyl bromide (2) and benzyl bromide (5). These NHC precursors were then treated with silver(I) acetate to yield the NHC,silver complexes [1,3-bis(4-methoxybenzyl)imidazol-2-ylidene]silver(I) acetate (4a), [4,5-dichloro-1,3-bis(4-methoxybenzyl)imidazol-2-ylidene]silver(I) acetate (4b), [1,3-bis(4-methoxybenzyl)benzimidazol-2-ylidene]silver(I) acetate (4c), (1,3-dibenzylimidazol-2-ylidene)silver(I) acetate (7a), (1,3-dibenzyl-4,5-dichloroimidazol-2-ylidene)silver(I) acetate (7b), and (1,3-dibenzylbenzimidazol-2-ylidene)silver(I) acetate (7c), respectively. The NHC precursor 3c, four NHC,silver complexes 4c and 7a,c were characterised by single-crystal X-ray diffraction method. The preliminary antibacterial activity of all the compounds was studied against Gram-negative bacteria Escherichia coli, and Gram-positive bacteria Staphylococcus aureus using the Kirby,Bauer disk-diffusion method. Almost all the NHC,silver complexes have shown high antibacterial activity compared to the NHC precursors. In addition, the NHC,silver complexes had their cytotoxicity investigated through MTT-based preliminary in vitro testing on the Caki-1 cell lines in order to determine their IC50 values. NHC,silver complexes 4a,c and 7a,c were found to have IC50 values of 7.3 (+/,6), 12.7(+/,3), 25.2 (+/,5), 2.5 (+/,3), 10.8 (+/,4) and 12.5 (+/,4) ,M respectively on the Caki-1 cell line. [source]

Cytotoxicity and sealing properties of four classes of endodontic sealers evaluated by succinic dehydrogenase activity and confocal laser scanning microscopy

EUROPEAN JOURNAL OF ORAL SCIENCES, Issue 2 2004
Serge Bouillaguet
The objectives of this study were to evaluate the cytotoxicity and sealing properties of four classes of endodontic sealers (PCS/Kerr, RoekoSeal/Roeko, TopSeal/Dentsply, and EndoREZ/Ultradent). For cytotoxicity testing (MTT method), the materials were either placed immediately in contact with cultured cells or 24 h after setting, then evaluated at three subsequent time points (24 h, 48 h, or 1 wk). For the leakage study, extracted human roots were obturated with acrylic cones and sealers and immersed for 48 h into rhodamine-labeled lipopolysaccharide. The roots were then observed under a confocal laser scanning microscope to estimate (semiquantitatively) the presence of the rhodamine-lipopolysaccharide (LPS) inside the canal. The results showed that cytotoxicity generally increased with time, and that most materials pose significant cytotoxic risks, particularly in the freshly mixed condition. Further, all materials showed significant leakage although there was large variation among teeth. Overall, the silicon-based material (Roeko Seal) was less cytotoxic and more effective in sealing root canals against LPS leakage than other materials. [source]

Cytotoxicity and Cell Cycle Effects of Bare and Poly(vinyl alcohol)-Coated Iron Oxide Nanoparticles in Mouse Fibroblasts

ADVANCED ENGINEERING MATERIALS, Issue 12 2009
Morteza Mahmoudi
Super-paramagnetic iron oxide nanoparticles (SPIONs) are recognized as powerful biocompatible materials for use in various biomedical applications, such as drug delivery, magnetic-resonance imaging, cell/protein separation, hyperthermia and transfection. This study investigates the impact of high concentrations of SPIONs on cytotoxicity and cell-cycle effects. The interactions of surface-saturated (via interactions with cell medium) bare SPIONs and those coated with poly(vinyl alcohol) (PVA) with adhesive mouse fibroblast cells (L929) are investigated using an MTT assay. The two SPION formulations are synthesized using a co-precipitation method. The bare and coated magnetic nanoparticles with passivated surfaces both result in changes in cell morphology, possibly due to clustering through their magnetostatic effect. At concentrations ranging up to 80,×,10,3,M, cells exposed to the PVA-coated nanoparticles demonstrate high cell viability without necrosis and apoptosis. In contrast, significant apoptosis is observed in cells exposed to bare SPIONs at a concentration of 80,×,10,3,M. Nanoparticle exposure (20,80,×,10,3,M) leads to variations in both apoptosis and cell cycle, possibly due to irreversible DNA damage and repair of oxidative DNA lesions, respectively. Additionally, the formation of vacuoles within the cells and granular cells indicates autophagy cell death rather than either apoptosis or necrosis. [source]

Labeling of Adipose-Derived Stem Cells by Oleic-Acid-Modified Magnetic Nanoparticles

ADVANCED FUNCTIONAL MATERIALS, Issue 8 2009
Lian Cen
Abstract The in vivo tracking of adipose derived stem cells (ASCs) is of essential concern when they are used as seed cells in tissue engineering. This study explores the feasibility of using magnetic nanoparticles (MNs), a type of contrast agents in magnetic resonance imaging (MRI), to label ASCs such that the labeled ASCs could be tracked in vivo by MRI non-invasively and repeatedly. To do this, MNs of <10,nm surface-coated with oleic acid are synthesized via a high-temperature solution-phase reaction. Cytotoxicity of the as-synthesized MNs at concentrations up to 0.1,mg,mL,1 on 104,cells,mL,1 ASCs is evaluated by LDH release. Since only minor cytotoxicity is detected, the effects of the labeling technique on cellular behaviors and uptake by labeled cells are investigated. Cell proliferation and differentiation with and without MNs are compared. The results show that proliferation of ASCs (104,cells,mL,1) labeled by MNs (0.05,mg,mL,1) is significantly enhanced and dependent on the labeling time. The MNs are located in the vesicles within cytoplasm of ASCs. The cellular uptake reaches as high as ,180,pg/cell. Nevertheless, the labeled ASCs still maintained adipogenic and osteogenic differentiation. Hence, the feasibility of labeling ASCs by oleic acid coated MNs is ascertained and it was better to label the cells during their quiescent stage. The labeled ASCs can also be in vivo detected by MRI in a subcutaneous model in vivo. Further MRI tracking of the labeled ASCs in long-term follow-up would thus follow this current study. [source]

Lipoxin A4 inhibited hepatocyte growth factor-induced invasion of human hepatoma cells

HEPATOLOGY RESEARCH, Issue 9 2009
Xiao-Yan Zhou
Aim:, Inflammation is a critical component of tumor progression. Lipoxin A4 (LXA4) has been approved for potent anti-inflammatory properties. Recently, it was reported that LXA4 repressed the expression and activity of cyclooxygenase-2 (COX-2), which is essential for invasion. However, there are few reports dealing with its effects on cancer. To explore whether LXA4 regulate invasion, the effects of LXA4 and its receptor agonist BML-111 on hepatocyte growth factor (HGF)-induced invasion of hepatoma cells and the possible mechanisms were researched. Methods:, Lipoxin A4 receptor (ALX) expression in HepG2 cells were measured through reverse transcription polymerase chain reaction and western blot. Cytotoxicity of LXA4 and BML-111 to HepG2 cells was detected by MTT and (3H)-TdR incorporation assay. Cell migration and invasion assays were performed using a Boyden chemotaxis chamber. COX-2 expression was detected by real-time polymerase chain reaction and western blot, respectively. Moreover, the expressions of matrix metalloproteinases (MMP)-2, MMP-9, I,B, and nuclear factor-,B (NF-,B) p65 were observed via western blot, and NF-,B transcriptional activity was tested by transfections and luciferase activities assay. Results:, ALX expression was detected in HepG2 cells, and suitable concentrations of LXA4 and BML-111 had no cytotoxicity to cells. LXA4 and BML-111 inhibited HGF-induced migration and invasion; downregulated COX-2, MMP-2 and -9; restrained HGF-induced I,B, degradation, NF-,B translocation and the transcriptional activity of NF-,B in HepG2 cells. Furthermore, exogenous PGE2 could reverse the inhibitory effects of LXA4 also BML-111 on HGF-induced invasion and migration partially. Conclusion:, LXA4 inhibited HGF-induced invasion of HepG2 cells through NF-,B/COX-2 signaling pathway partially. [source]

Cytotoxicity and genotoxicity of sodium percarbonate: a comparison with bleaching agents commonly used in discoloured pulpless teeth

INTERNATIONAL ENDODONTIC JOURNAL, Issue 2 2010
M. R. Fernández
Fernández MR, Carvalho RV, Ogliari FA, Beira FA, Etges A, Bueno M. Cytotoxicity and genotoxicity of sodium percarbonate: a comparison with bleaching agents commonly used in discoloured pulpless teeth. International Endodontic Journal, 43, 102,108, 2010. Abstract Aim, To evaluate the cytotoxicity and genotoxicity of sodium percarbonate (SPC) in comparison with bleaching agents used on discoloured pulpless teeth. Methodology, The cytotoxicity and genotoxicity of bleaching agents were evaluated both in their pure form as well as at concentrations commonly used in clinical practice. Hydrogen peroxide (HP), carbamide peroxide (CP), sodium perborate (SP) and SPC were diluted in Dulbecco's modified Eagle's medium (DMEM) in series. To evaluate the cytotoxicity, the survival of 3T3/NIH mouse fibroblasts was measured photometrically using an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay after a 24 h-exposure period. Genotoxicity was indicated by micronuclei (MN) formation, and modification of the normal cell was analysed by light microscopy (400×). Statistical analysis was performed by one-way anova, followed by a multiple-comparison Tukey post hoc test (P < 0.05). Results, All groups exhibited a dose-dependent cytotoxicity. However, CP showed a similar cytotoxic effect when compared with DMEM-untreated control (UC) group. HP and SPC were significantly more cytotoxic than SP. The genotoxicity test showed that SPC and SP had an intermediate rate of MN frequency when compared with the UC group. The mean rate of MN frequency for HP was higher and statistically more significant than for the other groups tested. No difference was observed when CP and UC groups were compared. Conclusions, Sodium percarbonate showed cytotoxicity and genotoxicity similar to those of the other products tested. However, before SPC is used clinically, studies should be conducted to confirm its safety in vivo. [source]

Cytotoxicity of MTA and Portland cement on human ECV 304 endothelial cells

INTERNATIONAL ENDODONTIC JOURNAL, Issue 9 2005
G. De Deus
Abstract Aim, To evaluate the cytotoxic effects of two brands of mineral trioxide aggregate (MTA) (Pro-Root MTA® and MTA Angelus®) and Portland cement (PC) on the human ECV 304 endothelial cell line. Methodology, Endothelial ECV 304 cells were incubated at 37 °C in an atmosphere of 95% air, 5% carbon dioxide and 100% humidity for 7 days and grown in F12 medium supplemented with 10% fetal bovine serum with 50 ,g mL,1 of gentamicin sulphate. Effects of the materials on mitochondrial functions were measured by a colorimetric assay. At each experimental time interval (24, 48 and 72 h), a dimethyl-thiazol-diphenyl tetrazolium bromid assay was conducted to measure cell viability. All assays were repeated three times to ensure reproducibility. Results were expressed as average absorbance (A570\,nm) ± SD and the data were analysed statistically by one-way analysis of variance and the Bonferroni post-test. A P -value <0.05 was considered statistically significant. Results, No statistically significant difference was shown between any of the experimental materials (P > 0.05). Conclusions, The two brands of MTA analysed, as well as the PC, initially showed a similar elevated cytotoxic effect that decreased gradually with time allowing the cell culture to become reestablished. [source]

Cytotoxicity of substances leached or dissolved from pulp capping materials

INTERNATIONAL ENDODONTIC JOURNAL, Issue 8 2005
B. N. Cavalcanti
Abstract Aim, To evaluate the cytotoxic effects of substances leached or dissolved from pulp capping materials on human pulp fibroblasts. Methodology, The substances were applied to cell cultures in conditioned media. The experimental groups were: GI (control; n = 24) , cultures treated with fresh medium; GII (n = 24) , cultures treated with calcium hydroxide cement; GIII (n = 24) , cultures treated with adhesive resin and GIV (n = 24) , cultures treated with 37% orthophosphoric acid. The media were conditioned by placing the crude materials in contact with fresh culture medium for 1 h. The cytotoxicity analysis was performed using the Trypan blue dye exclusion assay at times of 0, 6, 12 and 24 h for cell viability assay, and at 1, 3, 5 and 7 days for survival assay. Data were treated by anova (P < 0.05) and Tukey's test (P < 0.05). Results, GI and II presented similar cell viability and cell growth. GIII and IV exhibited statistically significant lower percentages of cell viability: GIV only at the 0 h experimental time, whereas in GIII this viability markedly diminished reaching values of 10% by 12 h. Cell growth was impaired only in cultures of GIII. Conclusions, Substances dissolved from the adhesive system tested were cytotoxic for human dental pulp fibroblasts in culture, whilst substances leached from calcium hydroxide were biocompatible. [source]

Cytotoxicity of dentine-bonding agents on human pulp cells in vitro

INTERNATIONAL ENDODONTIC JOURNAL, Issue 11 2002
F.-M. Huang
No abstract is available for this article. [source]

Cyclooxygenase-2 inhibitor celecoxib augments chemotherapeutic drug-induced apoptosis by enhancing activation of caspase-3 and -9 in prostate cancer cells

INTERNATIONAL JOURNAL OF CANCER, Issue 3 2005
Devendra S. Dandekar
Abstract Many tumors constitutively express high levels of the inducible form of proinflammatory enzyme, cyclooxygenase-2 (COX-2). Increased COX-2 expression is associated with tumor cell resistance to many cytotoxic chemotherapy drugs. Furthermore, increased resistance to cytotoxic antitumor drugs is also known to be dependent on associated stromal cells in many tumors. We investigated whether prostate tumor-associated stromal cells, marrow-derived osteoblasts, affect cytotoxicity of 2 antitumor drugs, COL-3 and docetaxel (TXTR), and whether it is dependent on COX-2 activity. We further examined whether inhibiting the activity of COX-2 negate the stroma-induced decrease in drug sensitivity in tumor cells. COX-2-specific inhibitor celecoxib (CXB) was used to inhibit COX-2 activity and associated alteration in cell death signaling was investigated. Coculturing PC-3ML cells with osteoblasts decreased the cytotoxicity of the tested antitumor drugs and was associated with increased COX-2 activity in PC-3ML cells. A significant decrease in drug-induced PGE2 increase and an increase in cytotoxicity were observed when cells were treated with COL-3 or TXTR combined with CXB. Cytotoxicity of single or combination treatment increased apoptosis, which was associated with caspase-3 and -9 activation, PARP cleavage, increased BAD protein, but decreased protein levels of XIAP and BCL- xL. Oral administration of CXB (40 mg/kg) to mice with PC-3ML tumors for 42 days increased tumor latency, decreased tumor growth and enhanced tumor control with COL-3 or TXTR. Overall, a synergistic enhancement of antitumor activity in combination treatment was observed in vitro and an additive effect in vivo. These observations suggest a potential clinical use of combined dosing of COX-2 inhibitors and cytotoxic drugs at lower, nontoxic dose than currently used to treat advanced prostate cancer. © 2005 Wiley-Liss, Inc. [source]

Enzymatic oxidation products of spermine induce greater cytotoxic effects on human multidrug-resistant colon carcinoma cells (LoVo) than on their wild-type counterparts

INTERNATIONAL JOURNAL OF CANCER, Issue 1 2002
Annarica Calcabrini
Abstract The occurrence of resistance to cytotoxic agents in tumor cells, associated with several phenotypic alterations, is one of the major obstacles to successful anticancer chemotherapy. A new strategy to overcome MDR of human cancer cells was studied, using BSAO, which generates cytotoxic products from spermine, H2O2 and aldehyde(s). The involvement of these products in causing cytotoxicity was investigated in both drug-sensitive (LoVo WT) and drug-resistant (LoVo DX) colon adenocarcinoma cells. Evaluation of clonogenic cell survival showed that LoVo DX cells are more sensitive than LoVo WT cells. Fluorometric assay and treatments performed in the presence of catalase demonstrated that the cytotoxicity was due mainly to the presence of H2O2. Cytotoxicity was eliminated in the presence of both catalase and ALDH. Transmission electron microscopic observations showed more pronounced mitochondrial modifications in drug-resistant than in drug-sensitive cells. Mitochondrial functionality studies performed by flow cytometry after JC-1 labeling revealed basal hyperpolarization of the mitochondrial membrane in LoVo DX cells. After treatment with BSAO and spermine, earlier and higher mitochondrial membrane depolarization was found in LoVo DX cells than in drug-sensitive cells. In addition, higher basal ROS production in LoVo DX cells than in drug-sensitive cells was detected by flow-cytometric analysis, suggesting increased mitochondrial activity in drug-resistant cells. Our results support the hypothesis that mitochondrial functionality affects the sensitivity of cells to the cytotoxic enzymatic oxidation products of spermine, which might be promising anticancer agents, mainly against drug-resistant tumor cells. © 2002 Wiley-Liss, Inc. [source]

Cytotoxicity and apoptosis induction of some selected marine bacteria metabolites

JOURNAL OF APPLIED MICROBIOLOGY, Issue 6 2005
J. Lin
Abstract Aims:, To study the potential apoptosis effects of cytotoxic marine bacterial metabolites on human HeLa cell line. Methods and Results:, After HeLa cells were routinely cultured, tetrazolium-based colorimetric assay for cytotoxicity was performed to screen the marine bacteria extracts showing 12 strains active. To find the potential active strain with apoptosis mechanism, a battery of apoptosis assays, including AO/EB staining, TUNEL assay (terminal-deoxynucleotidyl transferase mediated nick end labelling), gel electrophoresis and flow cytometry, were used to determine whether apoptosis was involved in HeLa cell cytotoxicity of marine bacterial extracts. The results indicated that four strains could induce cell shrinkage, cell membrane blebbing, formation of apoptotic body and DNA fragmentation. Conclusions:, Crude extracts of 12 of 153 strains of marine bacteria showed cytotoxic effects with ID50 ranged from 77·20 to 199·84 ,g ml,1, in which eight strains of bacteria were associated bacteria. The metabolites in the strains of QD1-2, NJ6-3-1, NJ1-1-1 and SS6-4 were able to induce HeLa cells apoptosis. Furthermore, the assessment by flow cytometry indicated that the hypodiploid apoptotic cells increased in a time-dependent manner, suggesting that induced apoptosis occurred from 24 h to 48 h after the extracts treatment. Significance and Impact of the Study:, Our results suggested that the compounds from fermentation in these four marine bacterial strains could be candidates for developing apoptosis specific anti-tumour agents with lower toxicity. This study indicated that associated marine bacteria could be good source to find cytotoxic metabolites, and some cytotoxic marine bacterial metabolites could have apoptosis mechanisms. [source]

Comparison of cytotoxic and inflammatory responses of photoluminescent silicon nanoparticles with silicon micron-sized particles in RAW 264.7 macrophages

JOURNAL OF APPLIED TOXICOLOGY, Issue 1 2009
Jonghoon Choi
Abstract Photoluminescent silicon nanoparticles have a bright and stable fluorescence and are promising candidates for bio-imaging, cell staining and drug delivery. With increasing development of nanotechnology applications for biomedicine, an understanding of the potential toxicity of nanoparticles is needed to assess safety concerns for clinical applications. The objective of this study was to compare biological responses of silicon nanoparticles (SNs, 3 nm diameter) with silicon microparticles (SMs, ,100,3000 nm diameter) in cultured murine macrophages (RAW 264.7) using standard protocols for assessing cytotoxicity/cell viability and inflammatory responses developed for micron-sized particles. SNs and SMs were exposed to macrophages with and without addition of endotoxin lipopolysaccharide (LPS), a positive inducer of tumor necrosis factor-alpha (TNF- ,), interleukin 6 (IL-6), and nitric oxide (NO). Cytotoxicity was assayed using the dye exclusion and MTT assays. Cell supernatants were assayed for production TNF- ,, IL-6 and NO. SNs at concentrations ,20 µg ml,1 exhibited no cytotoxicity or inflammatory responses; however, SNs and SMs >20 and 200 µg ml,1, respectively, increased cytotoxicity compared with controls. SMs induced concentration-related increases in TNF- , and IL-6 production; in contrast, the production of these cytokines was shown to decrease with increasing concentrations of SNs. NO production was not induced by SNs or SMs alone. Fluorescence microscopy demonstrated that SNs were associated with the macrophages, either internalized or attached to cell membranes. In conclusion, evaluating differences in biological responses for nanoparticles compared with microparticles of the same material may help improve tests to assess biological responses of nanoparticles that may be used in biomedical applications. Copyright © 2008 John Wiley & Sons, Ltd. [source]

Coumarin A/AA induces apoptosis-like cell death in HeLa cells mediated by the release of apoptosis-inducing factor

JOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 4 2009
Carolina Álvarez-Delgado
Abstract It has been demonstrated that naturally occurring coumarins have strong biological activity against many cancer cell lines. In this study, we assessed the cytotoxicity induced by the naturally isolated coumarin A/AA in different cancer cell lines (HeLa, Calo, SW480, and SW620) and in normal peripheral-blood mononuclear cells (PBMCs). Cytotoxicity was evaluated using the MTT assay. The results demonstrate that coumarin A/AA was cytotoxic in the four cancer cell lines tested and importantly was significantly less toxic in PBMCs isolated from healthy donors. The most sensitive cancer cell line to coumarin A/AA treatment was Hela. Thus, the programmed cell death (PCD) mechanism induced by this coumarin was further studied in this cell line. DNA fragmentation, histomorphology, cell cycle phases, and subcellular distribution of PCD proteins were assessed. The results demonstrated that DNA fragmentation, but not significant cell cycle disruptions, was part of the PCD activated by coumarin A/AA. Interestingly, it was found that apoptosis-inducing factor (AIF), a proapoptotic protein of the mitochondrial intermembrane space, was released to the cytoplasm in treated cells as detected by the western blot analysis in subcellular fractions. Nevertheless, the active form of caspase-3 was not detected. The overall results indicate that coumarin A/AA induces a caspase-independent apoptotic-like cell death program in HeLa cells, mediated by the early release of AIF and suggest that this compound may be helpful in clinical oncology. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:263,272, 2009; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20288 [source]

Protective effect of n-3 polyunsaturated fatty acid on primary culture of rat hepatocytes

JOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY, Issue 11 2007
Ryoichi Sohma
Abstract Background and Aim:, Recently, we reported on the beneficial clinical effects of eicosapentaenoic acid (EPA) in patients with primary biliary cirrhosis (PBC) who were unresponsive to ursodeoxycholic acid (UDCA). In this study we examined the effect of EPA on rat hepatocytes in primary culture. Methods:, Hepatocytes were isolated from rat liver by perfusion of collagenase and cultured with or without EPA. Cell damage induced by chenodeoxycholic acid (CDCA) was assessed by WST-8 assay and lactate dehydrogenase (LDH) release. PGE2 and LTB4 concentrations in the culture medium were measured by enzyme-linked immunosorbent assay (ELISA). cDNA was made from total RNA that was extracted from hepatocytes, and TaqMan polymerase chain reaction (PCR) was performed to assess the expression of CuZn and Mn superoxide dismutase (SOD) mRNA. Results:, When rat hepatocytes were cultured in the presence of EPA, the damage caused by CDCA was significantly decreased compared with cells cultured without EPA. Cytotoxicity significantly decreased in the presence of EPA. Furthermore, SOD mRNA expression was increased by adding EPA. These findings indicated that EPA protects cells by scavenging superoxide radicals (,O2,) mediated by SOD production. Conclusion:, EPA has a direct protective effect on rat hepatocytes, which is in agreement with the clinical efficacy of EPA in PBC patients. [source]

Activation of extracellular signal-regulated kinases potentiates hemin toxicity in astrocyte cultures

JOURNAL OF NEUROCHEMISTRY, Issue 3 2001
Raymond F. Regan
Hemin is present in intracranial hematomas in high micromolar concentrations and is a potent, lipophilic oxidant. Growing evidence suggests that heme-mediated injury may contribute to the pathogenesis of CNS hemorrhage. Extracellular signal-regulated kinases (ERKs) are activated by oxidants in some cell types, and may alter cellular vulnerability to oxidative stress. In this study, the effect of hemin on ERK activation was investigated in cultured murine cortical astrocytes, and the consequence of this activation on cell viability was quantified. Hemin was rapidly taken up by astrocytes, and generated reactive oxygen species (ROS) within 30 min. Increased immunoreactivity of dually phosphorylated ERK1/2 was observed in hemin-treated cultures at 30,120 min, without change in total ERK. Surprisingly, ERK activation was not attenuated by concomitant treatment with antioxidants (U74500A or 1,10-phenanthroline) at concentrations that blocked ROS generation. Cell death commenced after 2 h of hemin exposure and was reduced by antioxidants and by the caspase inhibitor Z-VAD-FMK. Cytotoxicity was also attenuated by MEK inhibition with PD98059 or U0126 at concentrations that were sufficient to prevent ERK activation. Whereas the effect of Z-VAD-FMK on cell survival was transient, the effect of MEK inhibitors was long-lasting. MEK inhibitors had no effect on cellular hemin uptake or subsequent ROS generation. The present results suggest that hemin activates ERK in astrocytes via a mechanism that is independent of ROS generation. This activation sensitizes astrocytes to hemin-mediated oxidative injury. [source]

Astroglia growth retardation and increased microglia proliferation by lithium and ornithine decarboxylase inhibitor in rat cerebellar cultures: Cytotoxicity by combined lithium and polyamine inhibition,

JOURNAL OF NEUROSCIENCE RESEARCH, Issue 3 2007
Gad M. Gilad
Abstract Lithium, the most prevalent treatment for manic-depressive illness, might have a neuroprotective effect after brain injury. In culture, lithium can exert neurotoxic effects associated with reduction in polyamine synthesis but neuroprotective effects as cultured neurons mature. Cumulative evidence suggests that lithium may exert some of its effects on neurons indirectly, by initially acting on glial cells. We used rat cerebellar cultures to ascertain the effects of lithium on ornithine decarboxylase (ODC) activity, the enzyme catalyzing the first step in polyamine synthesis, and to compare effects of lithium with those of the ODC inhibitor ,-difluoromethylornithine (DFMO) on neuron survival and glial growth. Switching cultures from high (25 mM) to low (5 mM) KCl concentrations served as the traumatic neuronal insult. The results indicate the following. 1) Whereas high depolarizing KCl concentration enhances neuron survival, it inhibits astroglial growth. 2) Lithium (LiCl; 1,5 mM) enhances neuronal survival but inhibits astroglial growth. 3) Lithium treatment leads to reduced ODC activity. 4) DFMO enhances neuron survival but inhibits astroglial growth. 5) Lithium and DFMO lead to transformation of astroglia from epithelioid (flat) to process-bearing morphology and to increased numbers of microglia. 6) Combined lithium plus DFMO treatment is cytolethal to both neurons and glia in culture. In conclusion, lithium treatment results in growth retardation and altered cell morphology of cultured astroglia and increased microglia proliferation, and these effects may be associated with inhibition of polyamine synthesis. This implies that direct effects on astrocytes and microglia may contribute to the effects of lithium on neurons. © 2006 Wiley-Liss, Inc. [source]

Cytotoxic effects of gingival retraction cords on human gingival fibroblasts in vitro

JOURNAL OF ORAL REHABILITATION, Issue 4 2004
C.-M. Liu
summary, The objective of this study was to determine the cytocompatibility of three different extracts of gingival retraction cords and to compare the cytotoxic effect of these materials on human gingival fibroblasts. Gingival retraction cords impregnated with aluminium sulphate (Gingi-Aid), dl -adrenaline HCl (Gingi-Pak) and non-drug-impregnated cord (Gingi-Plain) were eluted with culture medium for 10 min and 24 h. Cytotoxicity was judged using a tetrazolium bromide reduction assay. Our data demonstrated that gingival retraction cords applied alone almost completely inhibited cell viability (P < 0·05). In addition, the results also showed that the eluates from aluminium sulphate-impregnated cord, dl -adrenaline HCl-impregnated cord and non-drug-impregnated cord were cytotoxic to primary human gingival fibroblast cultures (P < 0·05). The cell viability of incubation of gingival fibroblasts containing 10-min eluates of aluminium sulphate, dl -adrenaline HCl and non-drug-impregnated cord was 61, 21 and 70%, respectively. The cell viability of incubation of gingival fibroblasts containing 24 h eluates of aluminium sulphate, dl -adrenaline HCl and non-drug-impregnated cord was 68, 58 and 72%, respectively. It was found that dl -adrenaline HCl-impregnated gingival retraction cord was the most toxic gingival retraction cord among the materials tested in all cultures (P < 0·05). The cytotoxicity decreased in an order of dl -adrenaline HCl-impregnated cord > aluminium sulphate-impregnated cord > non-drug-impregnated cord. The extent or degree of the cytotoxicity depended on the materials tested. Gingival retraction cords have significant potential for gingival toxicity. Careful management of gingiva retraction cords would lower the risk of potential gingival tissue damage during clinical application procedure and thus increase the success of prosthodontic procedures. [source]

A comparison of the in vitro cytotoxicity of two root canal sealers

JOURNAL OF ORAL REHABILITATION, Issue 4 2003
M. Dartar Öztan
summary, The purpose of this study was to compare the cytotoxicity of an epoxy resin-based sealer (AH Plus) and a silicone-based sealer (Roeko Seal Automix, RSA). Cytotoxicity was assessed using the MTT assay for mitochondrial enzyme activity and haemocytometer viable cell counting after 24, 48 and 72-h exposure to L929 cells. AH Plus and RSA showed no statistically significant difference in the degree of toxicity. Both sealers had a low toxic influence on the cells during the experimental period. This study indicates that epoxy resin-based sealer AH Plus and the silicone-based sealer RSA have similar levels of cytotoxicity to mouse fibroblasts. [source]

Long-term cytotoxicity of resin-based dental restorative materials

JOURNAL OF ORAL REHABILITATION, Issue 1 2002
S. Bouillaguet
Highly filled composites, Ormocers (organically modified ceramics) and ,smart' materials have been developed to overcome the polymerization shrinkage problems of conventional composite materials. The purpose of the current study was to investigate the effect of longer-term (up to 8 weeks) ageing of these resin-based dental restorative materials and determine the effect of post-curing on cytotoxicity. Twelve discs of each material (Colombus/IDR, Definite/Degussa, Ariston pHc/Vivadent) were either light-cured (Lc) or light-cured and post-cured (Pc). For cytotoxicity testing, the discs were placed in contact with cell culture medium (DMEM) and incubated at 37 °C. Extracts from composite materials were collected after 24 h and weekly over a time period of 8 weeks. Cytotoxicity of the eluates to cultured fibroblasts (Balb/c3T3) were measured by the succinic dehydrogenase (SDH) activity (MTT assay) and the results expressed in percentage of negative controls (Teflon discs). The results showed that ageing significantly influenced the cytotoxicity of the materials. Except for Ariston pHc, materials were less cytotoxic after 8 weeks of ageing than they were in early intervals and post-curing was not generally useful in reducing cytotoxicity. The Ariston pHc was initially moderately toxic, but then become highly cytotoxic for 5 weeks before returning to initial levels. The current study demonstrated the importance of assessing the cytotoxicity of resin composite materials at multiple times. [source]

Difference in susceptibilities of different cell lines to bilirubin damage

JOURNAL OF PAEDIATRICS AND CHILD HEALTH, Issue 1 2000
K-C Ngai
Objective: To investigate if there are differences in susceptibilities to bilirubin toxicity of different cell lines. Methodology: A modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method was adopted to study the cytotoxic effect of bilirubin on several commercially available cell lines including human glioblastoma (ATCC CRL 1690, T98G), human neuroblastoma (ATCC HTB-10, SK-N-MC), human liver (ATCC CCL 13, Chang Liver, HeLa markers) and a mouse fibroblast (ATCC CCL-1, NCTC Colon 929). Results: Cytotoxicity was observed when certain bilirubin:albumin molar ratios were exceeded in the medium of a cell line in culture. Different cells exhibited different susceptibilities to the cytotoxic effects of bilirubin; neuroblastoma and glioblastoma were most susceptible, fibroblasts were the least vulnerable. Conclusions: Our findings have confirmed the clinical impression that different cells sustain different degrees of cytotoxicities caused by bilirubin. [source]

Cytotoxicity and apoptosis enhancement in brain tumor cells upon coadministration of paclitaxel and ceramide in nanoemulsion formulations

JOURNAL OF PHARMACEUTICAL SCIENCES, Issue 7 2008
Ankita Desai
Abstract The objective of this study was to examine augmentation of therapeutic activity in human glioblastoma cells with combination of paclitaxel (PTX) and the apoptotic signaling molecule, C6 -ceramide (CER), when administered in novel oil-in-water nanoemulsions. The nanoemulsions were formulated with pine-nut oil, which has high concentrations of essential polyunsaturated fatty acid (PUFA). Drug-containing nanoemulsions were characterized for particle size, surface charge, and the particle morphology was examined with transmission electron microscopy (TEM). Epi-fluorescent microscopy was used to analyze nanoemulsion-encapsulated rhodamine-labeled PTX and NBD-labeled CER uptake and distribution in U-118 human glioblastoma cells. Cell viability was assessed with the MTS (formazan) assay, while apoptotic activity of PTX and CER was evaluated with caspase-3/7 activation and flow cytometry. Nanoemulsion formulations with the oil droplet size of approximately 200 nm in diameter were prepared with PTX, CER, and combination of the two agents. When administered to U-118 cells, significant enhancement in cytotoxicity was observed with combination of PTX and CER as compared to administration of individual agents. The increase in cytotoxicity correlated with enhancement in apoptotic activity in cells treated with combination of PTX and CER. The results of these studies show that oil-in-water nanoemulsions can be designed with combination therapy for enhancement of cytotoxic effect in brain tumor cells. In addition, PTX and CER can be used together to augment therapeutic activity, especially in aggressive tumor models such as glioblastoma. © 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 97:2745,2756, 2008 [source]

An extract of Lannea microcarpa: composition, activity and evaluation of cutaneous irritation in cell cultures and reconstituted human epidermis

JOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 7 2006
P. Picerno
Lannea microcarpa (Anacardiaceae) is a tropical tree used in African folk medicine and commercial dermopharmaceutical formulations. Fractionation and analysis of its polar extract allowed the identification of 4,-methoxy-myricetin 3- O -,- l -rhamnopyranoside, myricetin 3- O -,- l -rhamnopyranoside, myricetin 3- O -,- d -glucopyranoside, vitexin, isovitexin, gallic acid and epi-catechin, as the major constituents. In-vivo assay (the croton oil ear test in mice) showed that the extract had significant anti-inflammatory effect (ID50 = 900 ,g cm2) but ten times lower than that of indometacin (ID50 = 93 ,g cm2), the non-steroidal anti-inflammatory drug used as reference. Cytotoxicity and cutaneous irritation of the extract and its constituents were investigated. The crude extract and its major components did not affect cell viability in-vitro either in three different cultures (J774.A1, WEHI-164 and HEK-293) of cells grown in monolayers or in the reconstituted human epidermis (RHE, 3D model), nor did they cause release of pro-inflammatory mediators (IL-1,) or histomorphological modification of RHE. [source]

Effect of berberine on proliferation, cell cycle and apoptosis in HeLa and L1210 cells

JOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 8 2003
a Jantová
Previous studies on the anticancer activity of protoberberine alkaloids against a variety of cancer cell lines were extended to human tumour HeLa and murine leukemia L1210 cell lines. An attempt was also made to investigate the relationship between the cytotoxic activity of berberine and its molecular mechanism of action. Cytotoxicity was measured in-vitro using a primary biochemical screening according to Oyama and Eagle, and the growth inhibition assay. The in-vitro cytotoxic techniques were complemented by cell cycle analysis and determination of apoptotic DNA fragmentation in L1210 cells. Berberine acted cytotoxically on both tumour cell lines. The sensitivity of leukemia L1210 cells to the berberine was higher than that of HeLa cells. The IC100 was below 100 ,g mL,1 for HeLa cells and approached a 10 , mL,1 limit for the leukemia L1210 cells. For both cell lines the IC50 was found to be less than 4 ,g mL,1, a limit put forward by the National Cancer Institute (NCI) for classification of the compound as a potential anticancer drug. In L1210 cells treated with 10,50 , mL,1 berberine, G0/G1 cell cycle arrest was observed. Futhermore, a concentration-dependent decrease of cells in S phase and increase in G2/M phase was detected. In addition, apoptosis detected as sub-G0 cell population in cell cycle measurement was proved in 25,100 ,g mL,1 berberine-treated cells by monitoring the apoptotic DNA fragmentation (DNA ladder) using agarose gel electrophoresis. [source]

Fetal Ethanol Exposure Disrupts the Daily Rhythms of Splenic Granzyme B, IFN- ,, and NK Cell Cytotoxicity in Adulthood

ALCOHOLISM, Issue 6 2006
Alvaro Arjona
Background: Circadian (and daily) rhythms are physiological events that oscillate with a 24-hour period. Circadian disruptions may hamper the immune response against infection and cancer. Several immune mechanisms, such as natural killer (NK) cell function, follow a daily rhythm. Although ethanol is known to be a potent toxin for many systems in the developing fetus, including the immune system, the long-term effects of fetal ethanol exposure on circadian immune function have not been explored. Methods: Daily rhythms of cytotoxic factors (granzyme B and perforin), interferon- , (IFN- ,), and NK cell cytotoxic activity were determined in the spleens of adult male rats obtained from mothers who were fed during pregnancy with chow food or an ethanol-containing liquid diet or pair-fed an isocaloric liquid diet. Results: We found that adult rats exposed to ethanol during their fetal life showed a significant alteration in the physiological rhythms of granzyme B and IFN- , that was associated with decreased NK cell cytotoxic activity. Conclusion: These data suggest that fetal ethanol exposure causes a permanent alteration of specific immune rhythms that may in part underlie the immune impairment observed in children prenatally exposed to alcohol. [source]