INT Toxicity over Natural Bacterial Assemblages from Surface Oligotrophic Waters: Implications for the Assessment of Respiratory Activity.

Plankton community respiration (R) is a major component of the carbon flux in aquatic ecosystems. However, current methods to measure actual respiration from oxygen consumption at relevant spatial scales are not sensitive enough in oligotrophic environments where respiration rates are very low. To overcome this drawback, more sensitive indirect enzymatic approaches are commonly used as R proxies. The in vivo electron transport system (ETSvivo) assay, which measures the reduction of (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride salt, INT) to INT-formazan in the presence of natural substrate levels, was recently proposed as an indirect reliable estimation of R for natural plankton communities. However, under in vivo conditions, formazan salts could be toxic to the cells. Here, we test the toxicity of 0.2 mM of final INT concentration, widely used for ETSvivo assays, on natural bacterial assemblages collected in coastal and oceanic waters off Gran Canaria (Canary Islands, subtropical North Atlantic), in eight independent experiments. After 0.5 h of incubation, a significant but variable decline in cell viability (14-49%) was observed in all samples inoculated with INT. Moreover, INT also inhibited leucine uptake in less than 90 min of incubation. In the light of these results, we argue that enzymatic respiratory rates obtained with the ETSvivo method need to be interpreted with caution to derive R in oceanic regions where bacteria largely contribute to community respiration. Moreover, the variable toxicity on bacterial assemblages observed in our experiments questions the use of a single R/ETSvivo relationship as a universal proxy for regional studies.

INT reduction is a valid proxy for eukaryotic plankton respiration despite the inherent toxicity of INT and differences in cell wall structure.

The reduction of 2-para (iodophenyl)-3(nitrophenyl)-5(phenyl) tetrazolium chloride (INT) is increasingly being used as an indirect method to measure plankton respiration. Its greater sensitivity and shorter incubation time compared to the standard method of measuring the decrease in dissolved oxygen concentration, allows the determination of total and size-fractionated plankton respiration with higher precision and temporal resolution. However, there are still concerns as to the method's applicability due to the toxicity of INT and the potential differential effect of plankton cell wall composition on the diffusion of INT into the cell, and therefore on the rate of INT reduction. Working with cultures of 5 marine plankton (Thalassiosira pseudonana CCMP1080/5, Emiliania huxleyi RCC1217, Pleurochrysis carterae PLY-406, Scrippsiella sp. RCC1720 and Oxyrrhis marina CCMP1133/5) which have different cell wall compositions (silica frustule, presence/absence of calcite and cellulose plates), we demonstrate that INT does not have a toxic effect on oxygen consumption at short incubation times. There was no difference in the oxygen consumption of a culture to which INT had been added and that of a replicate culture without INT, for periods of time ranging from 1 to 7 hours. For four of the cultures (T. pseudonana CCMP1080/5, P. carterae PLY-406, E. huxleyi RCC1217, and O. marina CCMP1133/5) the log of the rates of dissolved oxygen consumption were linearly related to the log of the rates of INT reduction, and there was no significant difference between the regression lines for each culture (ANCOVA test, F = 1.696, df = 3, p = 0.18). Thus, INT reduction is not affected by the structure of the plankton cell wall and a single INT reduction to oxygen consumption conversion equation is appropriate for this range of eukaryotic plankton. These results further support the use of the INT technique as a valid proxy for marine plankton respiration.

Comparison of three different cell viability assays for evaluation of vanadyl sulphate cytotoxicity in a Chinese hamster ovary K1 cell line.

Previously, evaluation of sodium metavanadate (NaVO3) cytotoxicity after 24 h exposure of Chinese hamster ovary K1 (CHO-K1) cells revealed different sensitivity of the in vitro assays used starting from the neutral red (NR, 3-amino-7-dimethylamino-2-methylphenazine hydrochloride) test (detecting lysosomal and possibly the Golgi apparatus damage) as the most sensitive followed by the 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxyanilide inner salt (XTT) and resazurin (7-hydroxy-3H-phenoxazin-3-one-10-oxide) tests (mitochondrial disruption). The trypan blue (TB) staining (plasma membrane permeability) showed cytotoxicity of NaVO3 at a much higher NaVO3 concentration than the above-mentioned assays. In the current study, using the same experimental approach, we have assessed the toxicity of vanadyl sulphate (VOSO4) and compared the obtained results with NaVO3 action. Unlike metavanadate, VOSO4 treatment at 24 h resulted in similar sensitivity of the NR and resazurin tests. Nevertheless, following the 48-h incubation with VOSO4, the NR test showed markedly higher sensitivity than the resazurin test when comparing the half maximal inhibitory concentration values (61 and 110 µM for the NR and resazurin test, respectively, p < 0.05). The TB staining method was the least susceptible for detecting vanadyl cytotoxicity at each exposure time point. In summary, both the NR and resazurin tests can be advocated as similarly sensitive in detection of VOSO4-induced cytotoxicity in the CHO-K1 cell line at 24 h. However, the longer incubation time with VOSO4 showed that the NR test is more sensitive than the resazurin assay. The differences in the results between the cytotoxicity tests employed probably arise from dissimilar susceptibility of the endpoints (targets) measured with these tests to the damage by vanadium. Considering this, the current and the previous studies highlight the role of lysosomes (and possibly the Golgi apparatus) apart from mitochondria in the toxicity mechanism induced by inorganic vanadium in mammalian cells.

Phytochemical Content, Antioxidant and Cytotoxic Activities of Sedum spurium.

Sedum L. species are used for their hemostatic, antidiarrheal, antifungal, diuretic and wound healing properties, and there is growing interest in these species because of their usage in folk medicine. DPPH, SO, NO, and ABTS radical scavenging activities and protective effects against H(2)(02) induced cytotoxicity, as well as cytotoxic activities against the Hep-2 cell line of various extracts from Sedum spurium Bieb. were investigated. Besides, the total phenol, flavonoid, and flavonol contents of the extracts were determined to clarify their biological and phytochemical properties. Chromatographic studies on the most active extract led to the isolation of the major compound, identified as 2-methyl-erythritol by (1)H and (13)C NMR techniques. The EtOAc extract is found to be the most active extract in all tests. However, major compound of EtOAc extract did not possess tested activities. The EtOAC extract of S. spurium could be effective to improve antihemolytic defences of erythrocytes, and radical scavenging potential of the antioxidant mechanism. The extracts should be investigated in detail for their cytotoxic activities because of their possible pro-oxidant effects at high concentrations.

Avaliação multiparamétrica comparativa da citotoxicidade de diferentes materiais obturadores utilizados em pulpectomias em dentes decíduos através de modelo de simulação de canal radicular / Comparative multiparametric evaluation of the cytotoxicity of different obturator materials used in pulpectomies in deciduous teeth through a root canal simulation model

O objetivo do estudo foi avaliar a citotoxicidade de pastas obturadoras de canais radiculares de dentes decíduos através de um modelo de simulação de obturação de canal radicular de dente decíduo. A fim de orientar a escolha dos materiais, foi realizada uma revisão da literatura sobre as pastas utilizadas em estudos de Odontopediatria entre 2006 a 2016. Foram incluídos 21 artigos e concluiu-se que as pastas mais estudadas foram à base de óxido de zinco e eugenol (OZE) (n = 12), iodofórmio com hidróxido de cálcio (n = 8) e à base de hidróxido de cálcio (n = 3). Também foi executada uma revisão sistemática da literatura para a comparação dos resultados de efeito citotóxico (EC) de materiais obturadores de canais radiculares (MOCR) obtidos através de testes a base de sais de tetrazólio (TBST) e aqueles obtidos através de outro parâmetro de viabilidade celular nas mesmas células. O objetivo foi observar se os resultados de EC através das diferentes metodologias implicariam em tendências concordantes ou não de avaliação da citotoxicidade desses materiais. Foram incluídos 29 artigos e realizadas 279 comparações entre os resultados de avaliação do EC. Observou-se que 86,02% das comparações (n = 240) mostraram tendências similares de EC. Dentre os TBST, o MTT (n = 166) e o XTT (n = 81) apontaram tendências similares de EC em 87,95% e 92,59% das comparações. Os resultados sustentam a importância dos TBST para o rastreio da citotoxicidade de MOCR. Para a avaliação do EC das pastas selecionadas, foi desenvolvido um modelo de simulação de canal radicular que mimetizou as condições clínicas de obturação de canal radicular de dente decíduo limitando a exposição apical e a quantidade de material obturador usada Através do modelo foi realizada a avaliação multiparamétrica da citotoxicidade das pastas: à base de OZE, Vitapex®, Guedes-Pinto, pasta de Guedes, Calcicur®, Calen® espessada com óxido de zinco (Calen/OZ), pasta UFRJ e Endoflas® (n = 3) frente a células primárias semelhantes a osteoblastos humanos. O estudo foi executado através dos ensaios de redução do XTT, da incorporação do Vermelho neutro (VN) e da densidade de células aderidas pela eluição do corante Cristal violeta (CVDE). Ademais, as pastas foram avaliadas na proporção material: veículo da ISO 10993-5 (2009) através do teste do XTT (n = 5). Quando avaliadas pelo modelo e o teste do XTT, mostraram EC leve: Calcicur®, UFRJ, Endoflas®, Calen/OZ, Guedes-Pinto e à base de OZE. Vitapex® e Guedes apresentaram EC moderado e forte. Através do VN o EC foi leve para Calcicur®, Endoflas®, Calen®/OZ, Guedes-Pinto e à base de OZE; moderado para Vitapex® e UFRJ e forte para Guedes. Os resultados do CVDE foram similares aos do VN, com exceção da pasta Calen®/OZ, não citotóxica. Segundo a proporção da ISO, a pasta Calcicur® mostrou EC moderado e as demais EC forte. Concluiu-se que as pastas apresentaram resultados de EC menores quando avaliadas através do modelo proposto comparados aos obtidos com a proporção ISO. A exceção foi a pasta de Guedes pronta para uso considerada fortemente citotóxica em todas as avaliações (AU)

The aim of this study was to evaluate, comparatively, the cytotoxicity of different root canal filling pastes for primary teeth through a root canal simulating model. Aiming the selection of the pastes, a narrative literature review was performed with regard to the mostly evaluated root canal filling materials in Pediatric dentistry studies (2006-2016). In the review 21 papers were included and the most evaluated pastes were: zinc oxide and eugenol based (n = 11), iodoform with calcium hydroxide (n = 8) and calcium hydroxide-based (n = 3). Also a systematic review was peformed aiming to compare the cytotoxic effect (CE) results of root canal flling materials (RCFM), obtained through the tetrazolium salt-based tests (TSBT), and those obtained through other cell viability parameters assays. The objective was to observe if the results of CE through the different methodologies would imply in concordant or not trends of the cytotoxicity for such materials. We included 29 studies and performed 279 comparisons between the CE results. It was noted that 86.02% (n = 240) of such comparisons showed similar CE trends. Among the TSBT, MTT (n = 166) and XTT tests (n = 81) pointed to similar trends of CE in 87.95% and 92.59% of the comparisons. These review results support the value of the TSBT for the cytotoxicity screening of RCFM. Aiming the evaluation of CE of the selected pastes, a root canal simulating model was developed, which mimetized the clinical conditions of root canal filling for primary teeth through the controll of the the apical exposure and the amount of filling paste used. Through the model a multiparametric evaluation of the cytotoxicity was performed of the pastes for: zinc oxide and eugenol (ZOE) based, Vitapex®, Guedes-Pinto, Guedes, Calcicur®, Calen® thickened with zinc oxide (Calen/ZO), UFRJ paste and Endoflas® (n = 3) to human osteoblast-like primary cells. The study was performed through the assays of: the XTT reduction, the Neutral red (NR) up-take dye and adhered cell density evaluation through the Crystal violet dye elution (CVDE). Furthermore, the pastes were evaluated according to the material: vehicle ratio recommended by the ISO 10993-5 (2009) through the XTT reduction assay (n = 5). When the pastes were evaluated through the model and the XTT assay, a slight CE was observed for Calcicur®, UFRJ, Endoflas®, Calen/ZO, Guedes-Pinto and ZOE. Vitapex® and Guede showed moderate cytotoxic and strong CE. Through the NR the CE was slight for Calcicur®, Endoflas®, Calen/ZO, Guedes-Pinto and ZOE; moderate for Vitapex® and UFRJ and strong for Guedes. The CVDE results were similar to those of NR, with exception for the Calen/ZO, not cytotoxic. According to the ISO proportion, the Calcicur® showed moderate CE and the others strong CE. It was concluded that the CE results through the model were lower than those obtained through the ISO proportion. The exception was the ready-to-use Guedes paste, considered strongly cytotoxic in all evaluations (AU)

INT (2-(4-Iodophenyl)-3-(4-Nitrophenyl)-5-(Phenyl) Tetrazolium Chloride) Is Toxic to Prokaryote Cells Precluding Its Use with Whole Cells as a Proxy for In Vivo Respiration.

Prokaryote respiration is expected to be responsible for more than half of the community respiration in the ocean, but the lack of a practical method to measure the rate of prokaryote respiration in the open ocean resulted in very few published data leaving the role of organotrophic prokaryotes open to debate. Oxygen consumption rates of oceanic prokaryotes measured with current methods may be biased due to pre-incubation size filtration and long incubation times both of which can change the physiological and taxonomic profile of the sample during the incubation period. In vivo INT reduction has been used in terrestrial samples to estimate respiration rates, and recently, the method was introduced and applied in aquatic ecology. We measured oxygen consumption rates and in vivo INT reduction to formazan in cultures of marine bacterioplankton communities, Vibrio harveyi and the eukaryote Isochrysis galbana. For prokaryotes, we observed a decrease in oxygen consumption rates with increasing INT concentrations between 0.05 and 1 mM. Time series after 0.5 mM INT addition to prokaryote samples showed a burst of in vivo INT reduction to formazan and a rapid decline of oxygen consumption rates to zero within less than an hour. Our data for non-axenic eukaryote cultures suggest poisoning of the eukaryote. Prokaryotes are clearly poisoned by INT on time scales of less than 1 h, invalidating the interpretation of in vivo INT reduction to formazan as a proxy for oxygen consumption rates.

Characterization of polyethylene glycol-polyethyleneimine as a vector for alpha-synuclein siRNA delivery to PC12 cells for Parkinson's disease.

AIMS: Gene therapy targeting the SNCA gene yields promising results in the treatment of Parkinson's disease (PD). The most challenging issue of the RNAi gene therapy strategy is maintaining efficient delivery without inducing significant toxicity and other adverse effects. This study aimed to characterize polyethylene glycol-polyethyleneimine as a vector for alpha-synuclein siRNA delivery to PC12 cells for Parkinson's disease. METHODS: The characteristics of PEG-PEI/siSNCA were analyzed via gel retardation assay and assessments of particle size and zeta potential. MTT cytotoxicity assay and flow cytometry were used to detect cytotoxicity and transfection efficiency in PC12 cells. Confocal laser scanning microscopy was employed to examine the intracellular distribution of PEG-PEI/FITC-siSNCA after cellular uptake. RT-PCR and western blotting were used to measure SNCA expression. The MTT cytotoxicity assay was used to study the effect of PEG-PEI/siSNCA on cell viability. The protective effect of PEG-PEI/siSNCA on MPP+-induced apoptosis in PC12 cells was examined via flow cytometry and Hoechst staining. RESULTS: PEG-PEI/siSNCA complexes were well-developed; they exhibited appropriate particle sizes and zeta potentials at a mass ratio of 5:1. In vitro, PEG-PEI/siSNCA was associated with low cytotoxicity and high transfection efficiency. Complexes were capable of successfully delivering siSNCA into PC12 cells and releasing it from the endosome. Furthermore, PEG-PEI/siSNCA could effectively suppress SNCA mRNA expression and protected cells from death via apoptosis induced by MPP(+) . CONCLUSIONS: Our results demonstrate that PEG-PEI performs well as a vector for alpha-synuclein siRNA delivery into PC12 cells. Additionally, PEG-PEI/siSNCA complexes were suggested to be able to protect cells from death via apoptosis induced by MPP(+) . These findings suggest that PEG-PEI/siSNCA nanoparticles exhibit remarkable potential as a gene delivery system for Parkinson's disease.

Diminution of 2,3,5-triphenyltetrazolium chloride toxicity on Listeria monocytogenes growth by iron source addition to the culture medium.

Tetrazolium salts (TTZ) such as 2,3,5-triphenyltetrazolium chloride (TTC) are readily reduced by bacterial populations of various genus. The reduced form of these redox indicators is conspicuously colored allowing a quick and easy detection of growth. The studies are mainly confined to Gram negative bacteria because of an important toxic effect of tetrazolium salts on Gram positive bacteria. Indeed, we observed an important impact of different tetrazolium salts on Listeria monocytogenes growth, curiously limited to an increase in the duration of the lag phase. In this study, we demonstrate that increasing the iron concentration in a medium containing TTC leads to a significant decrease of the lag phase. L. monocytogenes growth was kinetically measured and growth parameters were estimated using the Baranyi model. While lag phase diminution was found to be iron concentration dependent, growth rate was not affected. Addition of iron enables growth of some strains totally inhibited by a 0.4 g/l of TTC and for the other a significant reduction of the latency is observed. The nature of the mechanism resulting in a decrease of the observed lag phase remains unclear. Then, the use of iron supplementation may be proposed to overcome the inhibitory effect of TTC on L. monocytogenes.

Exocytosis of MTT formazan could exacerbate cell injury.

MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] method is one of the most widely used methods to analyze cell proliferation and viability. It is taken up through endocytosis and is reduced by mitochondrial enzymes as well as endosomal/lysosomal compartments, then is transported to cell surfaces to form needle-like MTT formazans; however the effect of MTT itself still remains elusive. Our objective was to investigate the direct effects of MTT on in vitro SH-SY5Y cells. Results showed that the endocytosis of MTT did not cause obvious lesion and induce cell death, but the metabolism and exocytosis of MTT could dramatically damage cells. Our results also indicated that MTT could activate apoptosis related factors such as caspase-8, caspase-3 or accelerate the leakage of cell contents after the appearance of MTT formazan crystals. The present data suggest MTT method should be carefully chosen; otherwise the cell viability would be underestimated and incomparable.

MTT assay for cell viability: Intracellular localization of the formazan product is in lipid droplets.

Although MTT is widely used to assess cytotoxicity and cell viability, the precise localization of its reduced formazan product is still unclear. In the present study the localization of MTT formazan was studied by direct microscopic observation of living HeLa cells and by colocalization analysis with organelle-selective fluorescent probes. MTT formazan granules did not colocalize with mitochondria as revealed by rhodamine 123 labeling or autofluorescence. Likewise, no colocalization was observed between MTT formazan granules and lysosomes labeled by neutral red. Taking into account the lipophilic character and lipid solubility of MTT formazan, an evaluation of the MTT reaction was performed after treatment of cells with sunflower oil emulsions to induce a massive occurrence of lipid droplets. Under this condition, lipid droplets revealed a large amount of MTT formazan deposits. Kinetic studies on the viability of MTT-treated cells showed no harmful effects at short times. Quantitative structure-activity relations (QSAR) models were used to predict and explain the localization of both the MTT tetrazolium salt and its formazan product. These predictions were in agreement with experimental observations on the accumulation of MTT formazan product in lipid droplets.

Development of a naturally miniaturised testing method based on the mitochondrial activity of fern spores: a new higher plant bioassay.

One of the main concerns of current environmental toxicology is the low number of taxa used for standard bioassays. Ferns, with more than 10,000 living species, are the second largest group of vascular plants and are important components of numerous plant communities. Fern spores and gametophytes have long been recognized as useful models for plant research since they constitute a naturally miniaturised and economic higher plant model. Mitochondria are the main energy source in eukaryotic cells and any toxic damage will affect the whole organism. The reduction of tetrazolium salts to water-insoluble coloured formazan salts by the respiratory chain has been used for more than 50 years as a measure of cell mitochondrial activity and viability in eukaryotic organisms. Here, the reduction of 2,3,5-triphenyltetrazolium chloride (TTC) by mitochondria is adapted and optimized to measure fern spore or gametophyte viability. Procedures selected as optimum in the model species Dryopteris guanchica are as follows: bleach sterilization, incubation without shaking at 20 degrees C in the dark for 1-4h with 0.05-1.5% TTC in Dyer medium supplemented with 0.001-0.005% Tween 20 at pH 8. We conclude that this method constitutes a promising low cost bioassay for higher plant toxicity during development.

Hydrogen sulfide inhibits MPP(+)-induced apoptosis in PC12 cells.

AIMS: Hydrogen sulfide (H2S) is a well-known cytotoxic gas. Recently it has been shown to protect neurons against oxidative stress caused by glutamate, hypochlorous acid (HOCl), and beta-amyloid. The aim of the present study is to explore the cytoprotection of H2S against 1-methyl-4-phenylpyridinium ion (MPP(+))-induced apoptosis and the molecular mechanisms underlying in PC12 cells, a rat cell line derived from pheochromocytoma cells. MAIN METHODS: Cell viability was determined by the conventional 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay. Apoptosis was assessed by Hoechst 33258 nuclear staining and flow cytometric (FCM) analysis after propidium iodide staining. The mitochondrial membrane potential (MMP) was measured by rhodamine 123 (Rh123) probe and reactive oxygen species (ROS) were measured by dihydrorhodamine probe using FCM analysis. KEY FINDINGS: MPP(+) reduced the cell viability and induced apoptosis of PC12 cells along with dissipation of MMP as well as overproduction of ROS. Sodium hydrosulfide (NaHS), a H2S donor, protected PC12 cells against MPP(+)-induced cytotoxicity and apoptosis not only by reducing the loss of MMP, but also by attenuating an increase in intracellular ROS. SIGNIFICANCE: H2S significantly protected PC12 cells against cytotoxicity and apoptosis induced by MPP(+), which was associated with the inhibition by H(2)S of MPP(+)-induced dissipation of MMP and overproduction of ROS. These findings can significantly advance therapeutic approaches to the neurodegenerative diseases which are associated with oxidative stress, such as Parkinson's disease.

Evaluation of the toxicity of oligonucleotide/cationic nanoemulsion complexes on Hep G2 cells through MTT assay.

The purpose of this study was to evaluate the toxicity of the oligonucleotide/cationic nanoemulsion complexes on Hep G2 cells through MTT assay. Complexes exhibit droplet size, zeta potential and viscosity of approximately 270 nm, +50mV, and 1.0 cP. Different parameters which may have an influence on toxicity results obtained by MTT assay, i.e. cells number, concentration of MTT reagent and the addition of Soerensen's glycine buffer were first evaluated. In the optimized conditions (1 x 10(4) cells and 0.5 mg/mL MTT), the overall results showed that the addition of increasing amounts of complexes (or nanoemulsions) lead to a progressive toxicity on cells attributed to the presence of the cationic lipid stearylamine in the formulations, whatever the medias's pH is. The IC50 was approximately 200 microg/ml. Such results open interesting perspectives on the use of these nanoemulsions as oligonucleotide delivery systems for Hep G2 cells.

Prediction of acute toxicity in HPCT-1E3 hepatocytoma cells with liver-like transport activities.

A battery of in vitro methods has been developed for the prediction of acute oral toxicity, to reduce the number of animals used for this purpose. However, the results of these tests correlate more closely with lethal serum concentrations than with lethal doses. To address this issue, we have further evaluated the HPCT-1E3 model, which may be better able to emulate toxicokinetic factors that occur in vivo, due to the presence in these hepatocytoma cells of endogenous transmembrane carriers and a basal activity of xenobiotic metabolism. IC50 values produced by using the MTT test after a 48-hour incubation with 20 randomly-selected MEIC substances, correlated better with human oral LD50 values than with LC50 data, supporting this hypothesis. As with other models, the toxicity of receptor-specific rather than cytotoxic substances, for example digoxin, was underpredicted. When digoxin was removed from the correlation analysis, the coefficient of determination (r(2)) improved to 0.81, and none of remaining chemicals were wrongly predicted by more than one order of magnitude. IC50 values obtained with HepG2 cells under similar conditions (MEIC Test No. 3, 24 hours, MTT) correlated with human LD50 data with a r(2) value of 0.55. A direct comparison of HPCT-1E3 and HepG2 cells further suggested that the differences between them may be due to transport processes. In conclusion, the HPCT-1E3 model may be valuable in improving the prediction of lethal doses, rather than lethal serum concentrations.

The permeability and cytotoxicity of insulin-mimetic vanadium (III,IV,V)-dipicolinate complexes.

Vanadium (III,IV,V)-dipicolinate complexes with different redox properties were selected to investigate the structure-property relationship of insulin-mimetic vanadium complexes for membrane permeability and gastrointestinal (GI) stress-related toxicity using the Caco-2 cell monolayer model. The cytotoxicity of the vanadium complexes was assayed with 3-(4,5-dimethylthiazoyl-2-yl) 2,5-diphenyltetrazolium bromide (MTT) assays and the effect on monolayer integrity was measured by the trans-epithelial electric resistance (TEER). The three vanadium complexes exhibited intermediate membrane permeability (P(app) = 1.4-3.6x10(-6) cm/s) with low cellular accumulation level (<1%). The permeability of all compounds was independent of the concentration of vanadium complexes and excess picolinate ligands. Both V(III) and V(V)-dipicolinate complexes induced 3-4-fold greater reactive oxygen and nitrogen species (RONS) production than the V(IV)-dipicolinate complex; while the vanadium (III)-dipicolinate was 3-fold less damaging to tight junction of the Caco-2 cell monolayer. Despite the differences in apparent permeability, cellular accumulation, and capacity to induce reactive oxygen and nitrogen species (RONS) levels, the three vanadium complexes exhibited similar cytotoxicity (IC50 = 1.7-1.9 mM). An ion pair reagent, tetrabutylammonium, increased the membrane apparent permeability by 4-fold for vanadium (III and IV)-dipicolinate complexes and 16-fold for vanadium (V)-dipicolinate as measured by decrease in TEER values. In addition, the ion pair reagent prevented damage to monolayer integrity. The three vanadium (III,IV,V)-dipicolinate complexes may pass through caco-2 monolayer via a passive diffusion mechanism. Our results suggest that formation of ion pairs may influence compound permeation and significantly reduce the required dose, and hence the GI toxicity of vanadium-dipicolinate complexes.

Anti-proliferative and apoptotic effects of celecoxib on human chronic myeloid leukemia in vitro.

Celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, is the only non-steroidal anti-inflammatory drug so far which has been approved by the FDA for adjuvant treatment of patients with familial adenomatous polyposis. The molecular mechanism responsible for the anti-cancer effects of celecoxib is not fully understood. There is little data on the potential role of COX-2 in lymphoma pathogenesis. In view of the reported induction of apoptosis in cancer cells by cyclooxygenase-2 inhibitors, the present study is undertaken to test the effect of celecoxib on human chronic myeloid leukemia cell line, K562 and other hematopoietic cancer cell lines like Jurkat (human T lymphocytes), HL60 (human promyelocytic leukemia) and U937 (human macrophage). Treatment of these cells with celecoxib (10-100 microM) dose-dependently, reduced cell growth with arrest of the cell cycle at G0/G1 phase and induction of apoptosis. Further mechanism of apoptosis induction was elucidated in detail in K562 cell line. Apoptosis was mediated by release of cytochrome c into the cytoplasm and cleavage of poly (ADP-ribose) polymerase-1 (PARP-1). This was followed by DNA fragmentation. The level of anti-apoptotic protein Bcl-2 was decreased without any change in the pro-apoptotic Bax. Celecoxib also inhibited NF-kB activation. Celecoxib thus potentiates apoptosis as shown by MTT assay, cytochrome c leakage, PARP cleavage, DNA fragmentation, Bcl-2 downregulation and possibly by inhibiting NF-kB activation.

Effects of extracts of commonly consumed food supplements and food fractions on the permeability of drugs across Caco-2 cell monolayers.

PURPOSE: Extracts made from berries, herbs, and various plant materials, which might possess a range of activities, are used as health promoting products. Because little is known about their effects on the absorption of co-administered drugs, the effects of some food supplements, Finnish berries, and herbs were studied on the permeability of some commonly used drugs. METHODS: The permeabilities of verapamil, metoprolol, ketoprofen, paracetamol, and furosemide were studied across Caco-2 cell monolayers with contemporaneously administered extracts from flax seed, purple loosestrife, and Scots pine bark; bilberries, cowberries, and raspberries; oregano, rosemary, and sage. Toxicological tests were conducted to determine cellular damage. RESULTS: The effects of extracts on drug permeabilities were generally minor. Flax seed decreased the permeability of all drugs except verapamil. Purple loosestrife and pine decreased verapamil and metoprolol permeability. Changes caused by berries were mainly pH-related. Rosemary and oregano enhanced furosemide permeability. CONCLUSIONS: Ingestion of extracts of herbs and berries studied are not expected to markedly change the permeabilities of highly permeable drugs. Harmful effects at sites of or during absorption are unlikely. However, if high doses of extracts are administered with low permeable drugs in vitro, effects on drug permeabilities could not be excluded. Use of such extracts should therefore be evaluated during continuous medication.

Metal-induced hormesis requires cPKC-dependent glucose transport and lowered respiration.

Previously, cytotoxicity studies using an 3-(4,5 dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT)-based in vitro toxicity assay found that low concentrations of mercuric, cadmium and cupric chloride (0.7, 1 and 3 pM, respectively) induced hormesis in McCoy cells after 24 h exposure. An investigation of the biochemical events required for the induction of this phenomenon revealed that hormesis was dependent on two simultaneous but independent events, namely, an 11-15% conventional protein kinase C (cPKC)-dependent increase in glucose uptake and a protein synthesis-dependent 19-23% drop in mitochondrial respiration. The inhibition of either event was sufficient to abolish hormesis for all three metal toxicants. Furthermore, an investigation of the energy status of cells prior to and during hormesis revealed an oscillating level of ATP production found to be in phase with mitochondrial respiration, independent of cPKC-activated glucose transport and found to coincide with a 16-20% drop in AMP-activated protein kinase activity. These findings suggest that hormesis is not a form of energy compensation but is most likely a reductive burst where an increase in glucose uptake together with a simultaneous reduction in oxygen consumption results in a significant increase in reduction equivalents, which may then be utilized by cells to counteract the effects of oxidative stress induced by heavy metal toxicants.

Cultured rat vascular smooth muscle cells are resistant to methylamine toxicity: no correlation to semicarbazide-sensitive amine oxidase.

Methylamine (MA), a component of serum and a metabolite of nicotine and certain insecticides and herbicides, is metabolized by semicarbazide-sensitive amine oxidase (SSAO). MA is toxic to cultured human umbilical vein and calf pulmonary artery endothelial cells. Endothelial cells, which do not exhibit endogenous SSAO activity, are exposed to SSAO circulating in serum. In contrast, vascular smooth muscle cells (VSMC) do exhibit innate SSAO activity both in vivo and in vitro. This property, together with the critical localization of VSMC within the arterial wall, led us to investigate the potential toxicity of MA to VSMC. Cultured rat VSMC were treated with MA (10-5 to 1 M). In some cultures, SSAO was selectively inhibited with semicarbazide or MDL-72145 [(E)-2-(3,4-dimethoxyphenyl)-3-fluoroallylamine]. Cytotoxicity was measured via MTT, vital dye exclusion, and clonogenic assays. MA proved to be toxic to VSMC only at relatively high concentrations (LC(50) of 0.1 M). The inhibition of SSAO with semicarbazide or MDL-72145 did not increase MA toxicity, suggesting that the production of formaldehyde via tissue-bound, SSAO-mediated MA metabolism does not play a role in the minimal toxicity observed in isolated rat VSMC. The omission of fetal calf serum (FCS), which contains high SSAO activity, from media similarly showed little effect on cytotoxicity. We conclude that VSMC--in contrast to previous results in endothelial cells--are relatively resistant to MA toxicity, and SSAO does not play a role in VSMC injury by MA.