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Phenothiazine derivatives are well-known anti-psychotic drugs that possess several biological activities including anticancer activity. Much research provides data about its anticancer activity against human glioblastoma cell lines. Unfortunately, to date in vitro studies analyzing the impact of phenothiazines on the viability of human astrocytes have not been performed. However, it is possible to find one study about the viability of neurons and neurons with glia after incubation with perphenazine. Thus, in this study we measured the viability of human astrocytes after 24-, 48-, and 72-hour incubation with perphenazine and prochlorperazine dimaleate using the WST-1 assay. The obtained results suggest that perphenazine is safer for human astrocytes than prochlorperazine dimaleate. Moreover, 24-hour incubation with perphenazine or prochlorperazine did not significantly reduce cellular viability. It is a very important finding since previously we proved that in similar concentrations both drugs reduce the viability of the human glioblastoma U-87 MG cell line by approximately 50%. Therefore, the results suggest that phenothiazines can be used in glioblastoma treatment in concentrations that do not impact human astrocyte viability.

Purpose: In the following work, we investigated the effect of matcha green tea extract (MTE) on MCF-7 breast cancer cell viability and estrogen receptor-beta expression (ERβ). Methods: MCF-7 cells were stimulated with MTE at concentrations of 5 and 10 µg/ml. Cell viability was assessed using a water-soluble tetrazolium assay (WST-1 assay) after an incubation time of 72 h. ERβ was quantified at gene level by real-time polymerase chain reaction (PCR). A western blot (WB) was carried out for the qualitative assessment of the expression behavior of on a protein level. Results: The WST-1 test showed a significant inhibition of viability in MFC-7 cells after 72 h at 10 µg/ml. The WB demonstrated a significant quantitative decrease of ERβ at protein level with MTE concentrations of 10 µg/ml. In contrast, the PCR did not result in significant downregulation of ERβ. Conclusion: MTE decreases the cell viability of MCF-7 cells and furthermore leads to a decrease of ERβ at protein level. [ABSTRACT FROM AUTHOR]

The association between dysbiotic microbiota biofilm and colon cancer has recently begun to attract attention. In the study, the apitherapeutic effects of bee products (honey, bee venom, royal jelly, pollen, perga and propolis) obtained from the endemic Yığılca ecotype of Apis mellifera anatoliaca were investigated. Antibiofilm activity were performed by microplate assay using crystal violet staining to measure adherent biofilm biomass of Escherichia coli capable of forming biofilms. Bee venom showed the highest inhibition effect (73.98%) at 50% concentration. Honey, perga and royal jelly reduced biofilm formation by >50% at all concentrations. The antiproliferation effect on the HCT116 colon cancer cell line was investigated with the water‑soluble tetrazolium salt‑1 assay. After 48 h of honey application at 50% concentration, cell proliferation decreased by 86.51%. The high cytotoxic effects of royal jelly and bee venom are also remarkable. Additionally, apoptotic pathway analysis was performed by ELISA using caspase 3, 8 and 9 enzyme-linked immunosorbent assay kits. All bee products induced a higher expression of caspase 9 compared with caspase 8. Natural products that upregulate caspase proteins are promising therapeutic targets for proliferative diseases. [ABSTRACT FROM AUTHOR]

Tuberculosis is a highly infectious ailment worldwide. The emergence of multi-drug resistance and serious adverse effects of anti-TB drugs have led to the continuous search of natural candidates. This study aimed to analyse the chemical profile of Vitex pinnata (VP) bark lipophilic extract using GC-MS also evaluating its anti-TB and cytotoxic activities. GC-MS revealed a total of 81 compounds which representing 86% identified compounds. In vitro anti-TB of VP lipophilic extract was evaluated using the Microplate Alamar Blue Assay which exhibited MIC value of 62.5 µg/mL. In vitro cytotoxicity was evaluated using Water Soluble formazan assay recording IC50 > 100 and 200 µg/mL using Vero and A-549 cell lines, respectively. In silico docking study was performed on the major identified compounds, n-nonane showed the most favourable binding affinity (ΔG) equals to −33.34 Kcal/mol. The results obtained herein unravelled the potential use of VP n-hexane extract as a natural anti-TB. [ABSTRACT FROM AUTHOR]

Simple Summary: The outcome of equine artificial inseminations can be predicted with a high level of accuracy—87.9% if analyzed pre-chilling, and 95% if analyzed post-chilling—by optimizing a fertility prediction algorithm for individual stallions in conjunction with the Samson™ system and the reduction of the probe WST-1. This study aimed to determine whether an analysis of stallion ejaculate could accurately predict the likelihood of pregnancy resulting from artificial insemination in mares. This study involved 46 inseminations of 41 mares, using 7 standardbred stallions over a 5-week period at an Australian pacing stud. Semen quality was assessed immediately after collection and again after chilling at ~5 °C for 24 h. The assessment involved evaluating ejaculate volume, sperm concentration, and motility parameters using an iSperm® Equine portable device. After the initial evaluation, a subpopulation of cells was subjected to a migration assay through a 5 µm polycarbonate filter within a Samson™ isolation chamber over a 15 min period. The cells were assessed for their concentration, motility parameters, and ability to reduce the membrane impermeant tetrazolium salt WST-1. The data, combined with the stallion and mare's ages, were used to predict the likelihood of pregnancy, as confirmed by rectal ultrasound sonography performed 14 days post ovulation. The criteria used to predict pregnancy were optimized for each individual stallion, resulting in an overall accuracy of 87.9% if analyzed pre-chilling and 95% if analyzed post-chilling. This study suggests that an analysis of stallion ejaculate can be used to predict the likelihood of pregnancy resulting from artificial insemination in mares with a high level of accuracy. [ABSTRACT FROM AUTHOR]

Attention-deficit hyperactivity disorder (ADHD) is a neurodevelopmental polygenic disorder that affects more than 5% of children and adolescents around the world. Genetic and environmental factors play important roles in ADHD etiology, which leads to a wide range of clinical outcomes and biological phenotypes across the population. Brain maturation delays of a 4-year lag are commonly found in patients, when compared to controls of the same age. Possible differences in cellular growth rates might reflect the clinical observations in ADHD patients. However, the cellular mechanisms are still not elucidated. To test this hypothesis, we analysed the proliferation of induced pluripotent stem cells (iPSCs) and neural stem cells (NSCs) derived from male children and adolescents diagnosed with ADHD and with genetic predisposition to it (assessed using polygenic risk scores), as well as their respective matched controls. In the current pilot study, it was noticeable that NSCs from the ADHD group proliferate less than controls, while no differences were seen at the iPSC developmental stage. Our results from two distinct proliferation methods indicate that the functional and structural delays found in patients might be associated with these in vitro phenotypic differences, but start at a distinct neurodevelopmental stage. These findings are the first ones in the field of disease modelling of ADHD and might be crucial to better understand the pathophysiology of this disorder. [ABSTRACT FROM AUTHOR]

Curcuma longa (tumeric) is listed in several therapeutic regimens used in the management of cancer. The present study investigated the potential cytotoxic effects of ethanol extract of Curcuma longa (turmeric) leaves on HepG2 (a human hepatocellular carcinoma cell line), and MCF-7 (a human mammary gland adenocarcinoma cell line) cells. The HepG2 and MCF-7 cells were exposed to different concentrations; 2, 0.2, 0.02, 0.002, 0.0002, 0.00002, 0.000002 μg/ml, of the ethanol extract of C. longa leaves. The Water-Soluble tetrazolium salt (WST-1) assay was used to estimate the cell viability of the cells after which the percentage proliferation was computed. The results of this study indicated that C. longa had cytotoxic effect on the different cell lines, caused a decrease in proliferation and it is more selective to breast cancer cell lines based on the results of WST-1 analysis on the HepG2 and MCF-7 cells. These results suggest that C. longa leaf possesses cytotoxic properties, which can be exploited to develop novel cytotoxic drugs for cancer management. Keywords: Curcuma longa, turmeric, HepG2, MCF-7, WST-1, proliferation, cytotoxic, cancer

Purpose: In the following work, we investigated the nuclear peroxisome proliferator-activated receptor gamma (PPARγ)-dependent proliferation behavior of breast cancer cells after stimulation with matcha green tea extract (MTE). Methods: T47D cells were stimulated with MTE at concentrations of 5, 10 and 50 µg/ml. Cell viability was assessed using a WST-1 assay after an incubation time of 72 h. PPARγ expression was quantified at the gene level by real-time polymerase chain reaction (PCR). A western blot (WB) was carried out for the qualitative assessment of the expression behavior of on a protein level. Results: The WST-1 test showed a significant inhibition of viability in T47D cells after 72 h at 5, 10 and 50 µg/ml. The PCR showed an overexpression of PPARγ in T47D cells in all concentrations. At the concentration of 50 µg/ml the expression was significantly increased (p < 0.05). The WB demonstrated a significant quantitative increase of PPARγ at protein level with MTE concentrations of 10 and 50 µg/ml. In addition, there was a negative correlation between the overexpression of PPAR γ and the inhibition of proliferation. Conclusion: MTE decreases the cell viability of T47D cells and furthermore leads to an overexpression of PPARγ on protein and mRNA level. [ABSTRACT FROM AUTHOR]

Assessment of specific activity of Russian and foreign-made filgrastim products by biological in vitro methods is performed using different types of dyes. It is important to choose one cell staining dye in order to align the procedure of filgrastim specific activity assessment using cell culture. The aim of this study was to perform comparative assessment of tetrazolium and resazurin dyes in tests determining filgrastim ability to activate proliferation of sensitive cells. Materials and methods: NFS-60 (mouse myelogenous leukemia) cell line, 2nd International Standard for Granulocyte Colony Stimulating Factor (IS), as well as МТТ, MTS, WST-1, and alamarBlue dyes were used in the study. Proliferative activity of cells was assessed in vitro. The level of cell proliferation was assessed by fluorescence or absorbance intensity. Origin Pro 9.1. and Microsoft Excel applications were used for statistical processing of the obtained results. Results: the paper compares characteristics of the most widely used dyes. It describes the procedure for choosing optimal test conditions for some of the studied dyes. The authors analysed the potential of some factors, such as duration of cell suspension incubation with IS and with a dye, composition of the lysis buffer (for MTT staining), and different readout modes, to influence the final results. Despite the fact that all the studied dyes gave reproducible dose–response curves under the given test conditions, 50% effective concentrations showed no statistically significant differences in tests with only three dyes: МТТ, MTS, and alamarBlue (р > 0.05). Conclusions: better reproducibility of results was obtained in tests using МТТ and alamarBlue. The test procedure using alamarBlue is easier to perform and less time-consuming, it does not include the cell lysis stage and does not require additional reagents, therefore this dye may be recommended for harmonisation of the test procedure to be elaborated for the Russian Pharmacopoeia.

Zinc oxide nanoparticles (ZnO NPs) are one of the most widely used mineral particles. These nanoparticles are biocompatible and safe which can be used in medical-related industries. In this study, ZnO NPs were synthesized through an aqueous extract of Prosopis farcta fruit. Biosynthesized nanoparticles were identified through Ultraviolet–visible (UV–Vis), Powder X-Ray Diffraction (PXRD), Field Emission Scanning Electron Microscopy (FESEM), Energy Dispersive X-ray analysis (EDX), and Transmission Electron Microscopy (TEM) methods. The results of electron imaging showed that biosynthesized nanoparticles are hexagonal in shape with range size between 40 and 50 nm. Electron spectroscopy results showed that the bandgap of biosynthesized nanoparticles is 3.9 eV. The results of antifungal assay against Candida albicans showed 32–64 and 128–512 µg/ml values for minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC), respectively. Cytotoxicity of nanoparticles against breast cancer cells (MCF7) was performed using WST-1 assay and its IC50 was obtained 500 μg/ml.

This study aimed to determine whether an analysis of stallion ejaculate could accurately predict the likelihood of pregnancy resulting from artificial insemination in mares. This study involved 46 inseminations of 41 mares, using 7 standardbred stallions over a 5-week period at an Australian pacing stud. Semen quality was assessed immediately after collection and again after chilling at ~5 °C for 24 h. The assessment involved evaluating ejaculate volume, sperm concentration, and motility parameters using an iSperm® Equine portable device. After the initial evaluation, a subpopulation of cells was subjected to a migration assay through a 5 µm polycarbonate filter within a Samson™ isolation chamber over a 15 min period. The cells were assessed for their concentration, motility parameters, and ability to reduce the membrane impermeant tetrazolium salt WST-1. The data, combined with the stallion and mare’s ages, were used to predict the likelihood of pregnancy, as confirmed by rectal ultrasound sonography performed 14 days post ovulation. The criteria used to predict pregnancy were optimized for each individual stallion, resulting in an overall accuracy of 87.9% if analyzed pre-chilling and 95% if analyzed post-chilling. This study suggests that an analysis of stallion ejaculate can be used to predict the likelihood of pregnancy resulting from artificial insemination in mares with a high level of accuracy.

The Ca2+-mediated S100 family protein S100A6 has a crucial task in various intracellular and extracellular activities thereby demonstrating a possible involvement in the advancement and development of malignant tumors. S100A6 has been found to associate with receptor for advanced glycation end products, RAGE, through its extracellular extension. This extension is famously identified as a prominent receptor for many S100 family associates. Additionally, S100A6 binds to S100B protein and forms a heterodimer. Thus, we consider the S100B protein to be a prospective drug molecule to obstruct the interacting regions amongst S100A6 and RAGE V domain. We applied the NMR spectroscopy method to locate the binding area amid the S100A6m (mutant S100A6, cysteine at 3rd position of S100A6 is replaced with serine, C3S) and S100B proteins. The 1H–15N HSQC NMR titrations revealed the probable requisite dynamics of S100A6m and S100B interfaces. Utilizing data from the NMR titrations as input parameters, we ran the HADDOCK program and created a S100A6m-S100B heterodimer complex. The obtained complex was then superimposed with the reported complex of S100A6m-RAGE V domain. This superimposition displayed the possibility of S100B to be a potential antagonist that can block the interface area of the S100A6m and the RAGE V domain. Moreover, an in vitro cancer model using SW480 cells in water-soluble tetrazolium-1 assay (WST-1) showed a noticeable change in the cell proliferation as an effect of these proteins. Our study indicates the possibility to develop a S100B-like competitor that could play a key role in the treatment of S100- and RAGE-mediated human diseases. • S100A6m and S100B protein interacts with each other. • S100A6m and S100B forms a heterodimer complex successfully. • S100B blocks the interaction between S100A6m and RAGE-V. • S100B interfering with the interaction of S100A6m and RAGE-V domain is demonstrated by the WST-1 assay in vitro using SW480 Cells. [ABSTRACT FROM AUTHOR]

Zinc oxide nanoparticles (ZnO NPs) are one of the most widely used mineral particles. These nanoparticles are biocompatible and safe which can be used in medical-related industries. In this study, ZnO NPs were synthesized through an aqueous extract of Prosopis farcta fruit. Biosynthesized nanoparticles were identified through Ultraviolet–visible (UV–Vis), Powder X-Ray Diffraction (PXRD), Field Emission Scanning Electron Microscopy (FESEM), Energy Dispersive X-ray analysis (EDX), and Transmission Electron Microscopy (TEM) methods. The results of electron imaging showed that biosynthesized nanoparticles are hexagonal in shape with range size between 40 and 50 nm. Electron spectroscopy results showed that the bandgap of biosynthesized nanoparticles is 3.9 eV. The results of antifungal assay against Candida albicans showed 32–64 and 128–512 µg/ml values for minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC), respectively. Cytotoxicity of nanoparticles against breast cancer cells (MCF7) was performed using WST-1 assay and its IC50 was obtained 500 μg/ml. [ABSTRACT FROM AUTHOR]

Vinca alkaloids are usually included in a combination of chemotherapy applications for curative therapies. The best known are vincristine (VCR) and vinblastine (VBL), which are derived from Catharanthus roseus (Apocynaceae ). The purpose of this study was to demonstrate the antiproliferative and apoptotic effects of VCR and VBL on multiple myeloma (MM) cells. Toward this aim, the WST -1 assay was performed for cell proliferation analyses and the expression levels of the apoptotic gene regions were determined using quantitative RT -PCR. According to the WST-1 results, 2 Vinca alkaloids had an anti-proliferative effect on each of the .M1v1 cell lines. The WST-1 results showed that at the applied concentrations, VBL was more effective than VCR on the studied .M1v1 cells. On the other hand, the RT-PCR results indicated that alkaloids have a positive effect on a significant number of apoptotic gene regions at the molecular level. These results show that VCR and VBL have variable positive effects over the apoptotic genes in terms of gene expressions. From our findings, it was deduced that VBL is more effective than VCR with regards to the inhibition of cell proliferation and it has the potential to be used instead of VCR in clinical approaches. Consequently, this study promotes further studies of the Vinca alkaloids and their mechanism of actions. [ABSTRACT FROM AUTHOR]

Abstract PNGases are crucial targets and valuable tools in analyzing asparagine-linked carbohydrate moieties (N-glycans) of glycoproteins. Activity tests of PNGases have been little improved since their discovery four decades ago, and still rely on observing deglycosylation patterns of glycoproteins or glycopeptides using SDS-PAGE or HPLC analysis. These techniques cannot be easily adapted for automated sampling and high-throughput procedures. Herein, we describe a PNGase activity assay which relies on the conversion of WST-1, a yellowish, water-soluble tetrazolium dye (sodium 2-(4-Iodophenyl)-3-(4-nitro-phenyl)-5-(2,4-disulfophenyl)-2H-tetrazolate), into a blue formazan dye. In this work, we showed that WST-1 could be reduced by N-glycans, which were enzymatically released from glycoprotein substrates. After optimization of the assay conditions, the robustness of the method was challenged by quantifying the activity of various PNGase isoforms at different purification stages using a microwell plate reader. Furthermore, the assay could be used to obtain steady-state kinetics of PNGase H+ wild-type and mutant variants, which showed significant differences in their enzymatic reaction rates. The simplicity and robustness of this method might be of benefit for the detection of PNGase activity in routine applications of large amounts of samples. Graphical abstract Image 1 Highlights • A colorimetric method was established and optimized for testing PNGase activity. • Released N-glycans could be quantified using a water-soluble formazan dye. • Kinetic parameters of PNGase variants were determined using this optimized method. [ABSTRACT FROM AUTHOR]

Three known triterpenoids, namely ursolic acid (1), and the 27-E- and 27-Z-p-coumaric esters of ursolic acid (compounds 2, 3), were isolated together with a new triterpene 2,3-seco-taraxer-14-en-2,3-lactone [pycanocarpine (4)] from an ethanolic extract of Pleiocarpa pycnantha leaves. The structure of 4 was unambiguously assigned using NMR, HREIMS and X-ray crystallography. The cytotoxic activities of the compounds were evaluated against HeLa, MCF-7, KMST-6 and HT-29 cells using the WST-1 assay. Ursolic acid (1) displayed potent cytotoxic activity against HeLa, HT-29 and MCF-7 cells with IC50 values of 10, 10 and 20 µM respectively. The new compound 4 and its hydrolysed derivative 5 were selectively cytotoxic to the breast cancer cell line, MCF-7 with IC50 values 20 and 10 µM respectively. This is the first report on isolation of a 2,3-seco-taraxerene derivative from the Apocynaceae family and cytotoxic activityof P. pycnantha constituents.

BACKGROUND: BrdU is a commonly used reagent in cell proliferation assays, and WST-1 measurement is widely used to detect cell viability. However, no previous study has formally reported the combination of the two assays, which may be used to detect the proliferation and viability simultaneously. In this study, we examined the effect of adding BrdU 2 h prior to the WST-1 assay and tried to test the possibility of the combined detection using rat airway smooth muscle cells. RESULTS: The WST-1 measurements obtained from the combined detection were consistent with those obtained from the separate detection, which suggested that the addition of BrdU 2 h prior to the WST-1 analysis did not affect the WST-1 results. The BrdU measurements obtained from the combined detection also demonstrated the same trend as that obtained from the separate detection, and dosages of 200, 400 and 800 ng/ml testing reagent significantly inhibited the proliferation of rat airway smooth muscle cells. CONCLUSIONS: Our study suggests that the BrdU and WST-1 measurements can be applied simultaneously without mutual interference, which may increase the efficacy and consistency of these measurements to a certain extent.

Forages are important components of dairy cattle rations but might harbor a plethora of mycotoxins. Ruminants are considered to be less susceptible to the adverse health effects of mycotoxins, mainly because the ruminal microflora degrades certain mycotoxins. Yet, impairment of the ruminal degradation capacity or high ruminal stability of toxins can entail that the intestinal epithelium is exposed to significant mycotoxin amounts. The aims of our study were to assess (i) the mycotoxin occurrence in maize silage and (ii) the cytotoxicity of relevant mycotoxins on bovine intestinal cells. In total, 158 maize silage samples were collected from European dairy cattle farms. LC-MS/MS-based analysis of 61 mycotoxins revealed the presence of emerging mycotoxins (e.g., emodin, culmorin, enniatin B1, enniatin B, and beauvericin) in more than 70% of samples. Among the regulated mycotoxins, deoxynivalenol and zearalenone were most frequently detected (67.7%). Overall, 87% of maize silages contained more than five mycotoxins. Using an in vitro model with calf small intestinal epithelial cells B, the cytotoxicity of deoxynivalenol, nivalenol, fumonisin B1 and enniatin B was evaluated (0−200 µM). Absolute IC50 values varied in dependence of employed assay and were 1.2−3.6 µM, 0.8−1.0 µM, 8.6−18.3 µM, and 4.0−6.7 µM for deoxynivalenol, nivalenol, fumonisin B1, and enniatin B, respectively. Results highlight the potential relevance of mycotoxins for bovine gut health, a previously neglected target in ruminants.

Extracellular acidification is an important feature of tumor microenvironments but has yet to be successfully exploited in cancer therapy. The reversal of the pH gradient across the plasma membrane in cells that regulate intracellular pH (pHi) has potential to drive the selective uptake of weak acids at low extracellular pH (pHe). Here, we investigate the dual targeting of low pHe and hypoxia, another key feature of tumor microenvironments. We prepared eight bioreductive prodrugs based on the benzotriazine di-oxide (BTO) nucleus by appending alkanoic or aminoalkanoic acid sidechains. The BTO acids showed modest selectivity for both low pHe (pH 6.5 versus 7.4, ratios 2 to 5-fold) and anoxia (ratios 2 to 8-fold) in SiHa and FaDu cell cultures. Related neutral BTOs were not selective for acidosis, but had greater cytotoxic potency and hypoxic selectivity than the BTO acids. Investigation of the uptake and metabolism of representative BTO acids confirmed enhanced uptake at low pHe, but lower intracellular concentrations than expected for passive diffusion. Further, the modulation of intracellular reductase activity and competition by the cell-excluded electron acceptor WST-1 suggests that the majority of metabolic reductions of BTO acids occur at the cell surface, compromising the engagement of the resulting free radicals with intracellular targets. Thus, the present study provides support for designing bioreductive prodrugs that exploit pH-dependent partitioning, suggesting, however, that that the approach should be applied to prodrugs with obligate intracellular activation.

BACKGROUND: Tetrazolium-based assays are optimized to assess proliferation/toxicity of monolayer or suspension cells in microtiter plates. With regard to tissue engineering and regenerative medicine the need for in vivo like 3D microtissues has an increasing relevance. Applying tetrazolium-based assays to 3D culture systems is technically more challenging. The composed microenvironment may influence the assay standards, e.g. equal distribution of tetrazolium. OBJECTIVE: Evaluation of membrane-impermeable tetrazolium salt-based assays with regard to spheroid culture (3D) of human chondrocytes. METHODS:Chondrocytes were isolated from human articular cartilage.XTT,WST-1, andWST-8 were applied to monolayer cells (2D, varying cell numbers) and spheroids (3D, different sizes) in 96well plates. Formazan formation was measured spectrophotometrically after different incubation periods. Evaluation was done using phase contrast microsopy (toxicity), analyzing the correlation of cell number and absorbance signals (Gompertz function), and document signal over background ratio. RESULTS: In monolayer culture the assays showed a correlation between seeded cell numbers and absorption data. Spheroid sizes are directly related to the starting cell number. A correlation between size and absorbance was only detectable starting from 10,000 cells/aggregate. Phase contrast microscopy of monolayer cells revealed strong toxicity effects of the WST-1 (4 h) and XTT (8 h) assay and no signs of toxicity using WST-8. CONCLUSION: The WST-8 assay is non-toxic and revealed the highest sensitivity in comparison to the XTT or WST-1 assay. There is evidence, that only cells of the outer rim of spheroids are able to convert membrane-impermeable tetrazolium salts to formazans. [ABSTRACT FROM AUTHOR]

Trans-plasma membrane electron transport (tPMET) and the antioxidant roles of ascorbate reportedly play a role in protection of cells from damage by reactive oxygen species, which have been implicated in causing metabolic dysfunction such as insulin resistance. Skeletal muscle comprises the largest whole-body organ fraction suggesting a potential role of tPMET and ascorbate export as a major source of extracellular antioxidant. We hypothesized that skeletal muscle is capable of tPMET and ascorbate efflux. To measure these processes, we assayed the ability of cultured muscle cells, satellite cells, and isolated extensor digitorum longus (EDL) and soleus (SOL) to reduce two extracellular electron acceptors, water soluble tetrazolium salt 1 (WST-1), and dichlorophenolindophenol (DPIP). Ascorbate oxidase (AO) was utilized to determine which portion of WST-1 reduction was dependent on ascorbate efflux. We found that muscle cells can reduce extracellular electron acceptors. In C2C12 myotubes and satellite cells, a substantial portion of this reduction was dependent on ascorbate. In myotubes, glucose transporter 1 (GLUT1) inhibitors along with a pan-GLUT inhibitor suppressed tPMET and ascorbate efflux, while a GLUT4 inhibitor had no effect. The adenosine 5'-monophosphate (AMP)-activated protein kinase activator 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR) suppressed both tPMET and ascorbate efflux by myotubes, while insulin had no effect. Taken together, our data suggest that muscle cells are capable of tPMET and ascorbate efflux supported by GLUT1, thus illustrating a model in which resting muscle exports electrons and antioxidant to the extracellular environment. [ABSTRACT FROM AUTHOR]

Graphene and its derivative, because of their unique physical, electrical and chemical properties, are an important class of nanomaterials being proposed as foundational materials in nanomedicine as well as for a variety of industrial applications. A major limitation for graphene, when used in biomedical applications, is its poor solubility due to its rather hydrophobic nature. Therefore, chemical functionalities are commonly introduced to alter both its surface chemistry and biochemical activity. Here, we show that surface chemistry plays a major role in the toxicological profile of the graphene structures. To demonstrate this, we chemically increased the oxidation level of the pristine graphene and compared the corresponding toxicological effects along with those for the graphene oxide. X-ray photoelectron spectroscopy revealed that pristine graphene had the lowest amount of surface oxygen, while graphene oxide had the highest at 2.5% and 31%, respectively. Low and high oxygen functionalized graphene samples were found to have 6.6% and 24% surface oxygen, respectively. Our results showed a dose-dependent trend in the cytotoxicity profile, where pristine graphene was the most cytotoxic, with decreasing toxicity observed with increasing oxygen content. Increased surface oxygen also played a role in nanomaterial dispersion in water or cell culture medium over longer periods. It is likely that higher dispersity might result in graphene entering into cells as individual flakes ~1 nm thick rather than as more cytotoxic aggregates. In conclusion, changes in graphene's surface chemistry resulted in altered solubility and toxicity, suggesting that a generalized toxicity profile would be rather misleading. Copyright © 2016 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

One of the most convenient methods for measuring superoxide dismutase activity is a plate assay using xanthine oxidase and the water soluble tetrazolium WST-1. For reliable results with WST-1, certain aspects of the procedure need to be adhered to. This article describes an appropriate protocol that minimizes sources of variability. [ABSTRACT FROM AUTHOR]

Gingival tissue around teeth can be contacted by flowable photocured resin agents during operative procedures. The cytotoxicity of resin bonding agents toward human oral epithelial cells cannot be neglected. This study evaluates the cytotoxicity of resin bonding agents toward human oral epithelial cells using three assay systems. Materials and methods: Nine commercial resin bonding agents (Clearfil Protect Bond, Clearfil SE Bond, Prime & Bond NT, Prisma Universal Bond 3, UniFil Bond, 3M Single Bond, CharmBond, Compobond, and ExciTE) and two resin monomers (methyl methacrylate and triethylene glycol dimethacrylate) were used. The detection methods for the cell survival rate included: (1) the CellTiter 96 AQueous One Solution Cell Proliferation Assay System, (2) PreMix WST-1 Cell Proliferation Assay System, and (3) CellTiter-Glo Luminescent Cell Viability Assay System. One-way analysis of variance and Scheffé test were used for the statistical analyses. Results: Most of the components of the uncured bonding agents had cytotoxicity at 0.1 vol% (survival rates, 21.4% ± 1.4% to 113.1% ± 19.1%), except for UniFil Bond Primer (survival rates, 101.8% ± 3.5% to 113.1% ± 19.1%). More severe cytotoxicity was found at the 1.0 vol% concentration (survival rates, 0.2% ± 0.02% to 147.3% ± 10.8%), except for the methyl methacrylate monomer (survival rates, 96.5% ± 1.3% to 147.3% ± 10.8%). In the post-cured group, most bonding agents had little or no cytotoxicity, except for 3M Single Bond (survival rates, 52.4% ± 7.9% to 97.4% ± 17.8%) and Compobond (survival rates, 29.1% ± 7.1% to 79.5% ± 6.7%). Conclusion: The same material detected by different assay systems may give different cytotoxic effects, and examination by three methods may provide more-objective results. Furthermore, since most of the bonding agent components detected by the three assay systems showed cytotoxicity before curing (and some components even showed cytotoxicity after curing), the application of bonding materials in the oral cavity should be very carefully performed.

Determination of cell quantity in three-dimensional culture systems, such as the fibroblast-populated collagen matrix, can be difficult secondary to the abundance of extracellular collagen. The WST-1 assay, which can quantitate cellular processes in monolayer culture, was tested as a measure of relative cell quantity in a three dimensional culture system, the fibroblast-populated collagen matrix. The assay had acceptable accuracy and precision, and was not affected by the extracellular matrix. It was concluded that the assay was a reasonable alternative to other measures of cell quantity in the fibroblast-populated collagen matrix.

Cell proliferation assays are basic and essential techniques for assessing cellular function. Various colorimetric assays, such as MTT-, WST-1-, and resazurin-based assays, are available; however, studies directly comparing the suitability of each method for immune cell proliferation are scarce. Thus, we aimed to determine the best reagent and its optimal conditions based on variables such as cell number range, stimulation dose, kinetics, and compatibility with the cell division assay using CFSE fluorescence dye which is able to directly monitor divided cells by flow cytometry. In the absence of stimulation, MTT solubilized with SDS (MTT-SDS) and resazurin appeared to accurately reflect the cell numbers in a linear fashion. On the other hand, WST-1 exhibited a higher stimulation index following strong stimulation, whereas MTT-SDS and resazurin exhibited a better sensitivity to weak stimulation. A longer duration for stimulation did not necessarily increase sensitivity. CFSE staining revealed incremental cell division in response to anti-CD3 antibody stimulation in a dose-dependent manner. The cell numbers indirectly estimated from cell division profiles were consistent with the dose-response curve in the absorbance of MTT-SDS and resazurin. The absorbance does not increase before cell division, irrespective of T cell activation status, suggesting that these reagents reflect the cell number but not the cellular volume. Collectively, resazurin and MTT-SDS seem to be more reliable than others, and thus appear applicable in various conditions for the immune cell experiments. [ABSTRACT FROM AUTHOR]

Trans-plasma membrane electron transport (tPMET) and the antioxidant roles of ascorbate reportedly play a role in protection of cells from damage by reactive oxygen species, which have been implicated in causing metabolic dysfunction such as insulin resistance. Skeletal muscle comprises the largest whole-body organ fraction suggesting a potential role of tPMET and ascorbate export as a major source of extracellular antioxidant. We hypothesized that skeletal muscle is capable of tPMET and ascorbate efflux. To measure these processes, we assayed the ability of cultured muscle cells, satellite cells, and isolated extensor digitorum longus (EDL) and soleus (SOL) to reduce two extracellular electron acceptors, water soluble tetrazolium salt 1 (WST-1), and dichlorophenolindophenol (DPIP). Ascorbate oxidase (AO) was utilized to determine which portion of WST-1 reduction was dependent on ascorbate efflux. We found that muscle cells can reduce extracellular electron acceptors. In C2C12 myotubes and satellite cells, a substantial portion of this reduction was dependent on ascorbate. In myotubes, glucose transporter 1 (GLUT1) inhibitors along with a pan-GLUT inhibitor suppressed tPMET and ascorbate efflux, while a GLUT4 inhibitor had no effect. The adenosine 5′-monophosphate (AMP)-activated protein kinase activator 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR) suppressed both tPMET and ascorbate efflux by myotubes, while insulin had no effect. Taken together, our data suggest that muscle cells are capable of tPMET and ascorbate efflux supported by GLUT1, thus illustrating a model in which resting muscle exports electrons and antioxidant to the extracellular environment.

The objective of this study was to assess the cytotoxicity of EndoSeal, a novel mineral trioxide aggregate-based root canal sealerin comparison with two commonly used sealers, AH Plus and Sealapex. For cytotoxicity assay, MG-63 cells and human gingivalfibroblasts were incubated in culture medium containing eluates of each sealer at 1, 3, and 7 days. Cell metabolism was evaluatedby the WST-1 assay. For cell adhesion assay, disc specimens were fabricated from EndoSeal and AH Plus. MG-63 cells and humangingival fibroblasts were seeded on the discs, and after 1 day and 7 days of incubation, cell morphology and cell adhesion wereexamined by SEM. The order of cytotoxicity of root canal sealers was as follows: EndoSeal < AH Plus < Sealapex. Both types of cellsseeded on the EndoSeal specimens were much larger, flat with rough margins compared to those on the AH Plus specimens. Theseresults suggest that EndoSeal has a satisfactory cytocompatibility. [ABSTRACT FROM AUTHOR]

The article discusses a research conducted to examine the antioxidative capacity of polygonum cuspidatum also known as Japanese knotweed by measuring its trans-resveratrol and glycoside content. Topics include use of 1,1 -diphenyl-2-picrylhydrazyl (DPPH) and water-soluble tetrazolium salt-1 (WST-1), and amount of resveratrol being affected due to seasonal, geographical and environmental factors. It also presents graph and tables depicting the observations of the analysis.

Three known triterpenoids, namely ursolic acid (1), and the 27-E- and 27-Z-pcoumaric esters of ursolic acid (compounds 2, 3), were isolated together with a new triterpene 2,3-seco-taraxer-14-en-2,3-lactone [pycanocarpine (4)] from an ethanolic extract of Pleiocarpa pycnantha leaves. The structure of 4 was unambiguously assigned using NMR, HREIMS and X-ray crystallography. The cytotoxic activities of the compounds were evaluated against HeLa, MCF-7, KMST-6 and HT-29 cells using the WST-1 assay. Ursolic acid (1) displayed potent cytotoxic activity against HeLa, HT-29 and MCF-7 cells with IC50 values of 10, 10 and 20 μM respectively. The new compound 4 and its hydrolysed derivative 5 were selectively cytotoxic to the breast cancer cell line, MCF-7 with IC50 values 20 and 10 μM respectively. This is the first report on isolation of a 2,3-seco-taraxerene derivative from the Apocynaceae family and cytotoxic activityof P. pycnantha constituents. [ABSTRACT FROM AUTHOR]

The introduction of new immunosuppressive drugs in the last two decades has been associated with a significant decline in the prevalence of acute rejection and a huge improvement of graft survival. Monitoring blood levels of immunosuppressive drugs is the most common way to control drug doses in renal transplant patients. This approach is useful and widely used but doesn't give accurate information about the immune status of the patient. For this goal, there are many “in house” protocols which give more information, but cannot be standardized, limiting their applicability to compare results between different laboratories. In this study we compare three classical functional methods to evaluate the immune response: Mixed lymphocyte reaction (MLR), phytohemagglutinin stimulated peripheral blood lymphocytes (PBLs), and anti-CD3 monoclonal antibodies (mAbs) against PBL with the only FDA-labeled assay to measure the patient immune status: Cylex ImmuKnow® that measures the intracelullar ATP in CD4+ lymphocytes. We used n=111 stable renal transplant patients, all the patients with more than one year functioning grafts. We referred the results to a control population of healthy blood donors (n=125). Results: Measurement of intracellular ATP in CD4+ lymphocytes is able to differentiate immunosuppressed populations in renal transplant patients from health controls (242.30±21.62 vs. 386.43±25.12, p 0.0001). By contrary, there were no differences between controls and renal recipients when functional response was measured by MLR, PHA and anti-CD3 mAbs (2.48±0.45 vs. 2.37±0.41; 2.84±0.76 vs. 2.37±0.32; 2.32±0.34 vs. 1.89±0.38 respectively). In summary, our results show that the measurement of ATP in CD4+ lymphocytes gives more accurate information in comparison to the classical methods. [ABSTRACT FROM AUTHOR]

Background Carbon nanotubes (CNT) can induce lung inflammation and fibrosis in rodents. Several studies have identified the capacity of CNT to stimulate the proliferation of fibroblasts. We developed and validated experimentally here a simple and rapid in vitro assay to evaluate the capacity of a nanomaterial to exert a direct pro-fibrotic effect on fibroblasts. Methods The activity of several multi-wall (MW)CNT samples (NM400, the crushed form of NM400 named NM400c, NM402 and MWCNTg 2400) and asbestos (crocidolite) was investigated in vitro and in vivo. The proliferative response to MWCNT was assessed on mouse primary lung fibroblasts, human fetal lung fibroblasts (HFL-1), mouse embryonic fibroblasts (BALB- 3T3) and mouse lung fibroblasts (MLg) by using different assays (cell counting, WST-1 assay and propidium iodide PI staining) and dispersion media (fetal bovine serum, FBS and bovine serum albumin, BSA). C57BL/6 mice were pharyngeally aspirated with the same materials and lung fibrosis was assessed after 2 months by histopathology, quantification of total collagen lung content and pro-fibrotic cytokines in broncho-alveolar lavage fluid (BALF). Results MWCNT (NM400 and NM402) directly stimulated fibroblast proliferation in vitro in a dosedependent manner and induced lung fibrosis in vivo. NM400 stimulated the proliferation of all tested fibroblast types, independently of FBS- or BSA- dispersion. Results obtained by WST1 cell activity were confirmed with cell counting and cell cycle (PI staining) assays. Crocidolite also stimulated fibroblast proliferation and induced pulmonary fibrosis, although to a lesser extent than NM400 and NM402. In contrast, shorter CNT (NM400c and MWCNTg 2400) did not induce any fibroblast proliferation or collagen accumulation in vivo, supporting the idea that CNT structure is an important parameter for inducing lung fibrosis. Conclusions In this study, an optimized proliferation assay using BSA as a dispersant, MLg cells as targets and an adaptation of WST-1 as readout was developed. The activity of MWCNT in this test strongly reflects their fibrotic activity in vivo, supporting the predictive value of this in vitro assay in terms of lung fibrosis potential [ABSTRACT FROM AUTHOR]

Highlights: [•] A novel method for measuring superoxide anion radical scavenging activity was established. [•] The method is simple, rapid and sensitive. [•] The O2 − scavenging activities of tissue homogenates, serum and herbal extracts were determined. [•] The method can be used for screening superoxide anion radical scavengers. [ABSTRACT FROM AUTHOR]

Background Combretastatin A-4 (CA-4), which is an excellent antineoplastic agent, was isolated from Combretum caffrum. To date, structural modification studies of CA-4 have focused predominantly on the construction of new therapeutic agents for drug discovery. As a part of our ongoing work towards the modification of natural products, we have focused on the 3'-Osubstituent groups in the B-ring of CA-4 under the hypothesis that these novel derivatives will possess good bioactivities and behave as effective antiproliferative pro-drugs. Results A series of novel CA-4 derivatives, which contained a 3'-O-substituted carbonic ether moiety, were synthesized and evaluated for their antitumor activities against four tumor cell lines, including MDA-MB-231, MCF-7, K562 and A549 cells. These derivatives exhibited clear antitumor activities, and CA-4E, in particular, showed the highest bioactivity of all of the derivatives tested against all four tumor cell lines, with IC50 values in the range of 1 to 180 nM. Based on its high bioactivity, CA-4E was subsequently selected to investigate the antitumor mechanism of these synthetic compounds. The cell cycle results demonstrated that CA-4E induced time- and dose-dependent G2/M arrest in a similar manner to CA-4, although its effect was more powerful than that of CA-4, and the apoptosis data showed that CA-4E induced cellular apoptosis in a dose-dependent manner. Conclusions The newly synthesized CA-4 derivatives exhibited good antitumor activities in vitro, with CA-4E, in particular, showing the highest bioactivity of all of the compounds tested. Furthermore, CA-4E induced time- and dose-dependent G2/M arrest and cellular apoptosis in a dose-dependent manner. Taken together, these results suggest that CA-4E should be subjected to further investigation as a potential anticancer drug candidate. [ABSTRACT FROM AUTHOR]

Friedreich ataxia (FRDA) is an autosomal recessive neuro- and cardiodegenerative disorder for which there are no proven effective treatments. FRDA is caused by decreased expression and/or function of the protein frataxin. Frataxin chaperones iron in the mitochondrial matrix for the assembly of iron–sulfur clusters (ISCs), which are prosthetic groups critical for the function of the Krebs cycle and the mitochondrial electron transport chain (ETC). Decreased expression of frataxin or the yeast frataxin orthologue, Yfh1p, is associated with decreased ISC assembly, mitochondrial iron accumulation, and increased oxidative stress, all of which contribute to mitochondrial dysfunction. Using yeast depleted of Yfh1p, a high-throughput screening (HTS) assay was developed in which mitochondrial function was monitored by reduction of the tetrazolium dye WST-1 in a growth medium with a respiration-only carbon source. Of 101 200 compounds screened, 302 were identified that effectively rescue mitochondrial function. To confirm activities in mammalian cells and begin understanding mechanisms of action, secondary screening assays were developed using murine C2C12 cells and yeast mutants lacking specific complexes of the ETC, respectively. The compounds identified in this study have potential relevance for other neurodegenerative disorders associated with mitochondrial dysfunction, such as Parkinson disease. [ABSTRACT FROM PUBLISHER]

Abstract: A specific colorimetric assay for the determination of glucose-6-phosphate (G6P) was developed. This assay is based on the oxidation of G6P in the presence of glucose-6-phosphate dehydrogenase (G6PD) and nicotinamide adenine dinucleotide phosphate (NADP+); the NADPH thereby generated reduces the tetrazolium salt WST-1 [2-(4-indophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H tetrazolium, monosodium salt] to water-soluble yellow-colored formazan with 1-methoxy-5-methylphenazium methylsulfate (1-mPMS) as an electron carrier. The assay is optimized for reaction buffer pH, enzyme/dye concentration, and reaction time course. The limit of detection of the assay is 0.15μM (15pmol/well). The usefulness of the assay is demonstrated by the accurate measurement of the G6P concentration in fetal bovine serum (FBS). [Copyright &y& Elsevier]

Abstract: The UVA-irradiation of 10J/cm2 on HaCaT keratinocytes increased 59.1% of the intracellular reactive oxygen species (ROS) by NBT assay and the cell viability decreased to 31.5% by WST-1 assay, comparing to the non-irradiated control. In the presence of fullerene-C60 (C60) incorporated in phospholipid membrane vehicle (LiposomeFullerene: Lpsm-Flln) of 250–500ppm, they were restored to −9.1% to +2.3% of the ROS and 83.0–84.8% of the cell viability, but scarcely restored by the liposome without C60 (Lpsm). In HaCaT cells administered with Lpsm-Flln (150ppm), C60 was ingested at the intracellular concentrations of 1.4–21.9ppm for 4–24h, and, intracellular C60 was excreted by 80% at 4h after rinsing-out, and decreased to 2–10% after 24–48h. C60 was predominantly distributed around the outside of nuclear membrane without deterioration of intact cell morphology according to fluorescent immunostain. Thus Lpsm-Flln is found to be an effective antioxidant that could preserve HaCaT keratinocytes against UVA-induced cellular injury. Lpsm-Flln has a potential to serve as a cosmetic material for skin protection against UVA. [Copyright &y& Elsevier]

Abstract: This study was conducted to characterize the production of superoxide ions by leukocytes in whole blood of the American alligator (Alligator mississippiensis). We used WST-1, a tetrazolium salt which can be reduced to a water-soluble formazan compound with high molar absorptivity at 438 nm, to probe the production of superoxide by alligator leukocytes. Incubation of alligator whole blood with WST-1 resulted in a time- and concentration-dependent increase in absorbance of the plasma at 438 nm. The reduction of WST-1 was inhibited in a concentration-dependent manner by superoxide dismutase, an enzyme that catalyzes the reduction of superoxide to peroxide, confirming that the reduction of WST-1 was due to the presence of superoxide. Treatment of whole blood with nitrotetrazolium blue (NBT) resulted in the staining of heterophils and monocytes, enforcing the idea that that the production of superoxide is due to the presence of leukocytes, and not other blood cell components. It is interesting to note that the production of superoxide by the alligator leukocytes required no external stimulation while human leukocytes must be stimulated with an immunological challenge before producing superoxide. This is the first report of the production of superoxide as an innate immune mechanism in crocodilians. [Copyright &y& Elsevier]

The effects of econazole, an antifungal drug applied for treatment of keratitis and mycotic corneal ulcer, on cytosolic-free Ca2 + concentrations ([Ca2 +]i) and viability of corneal cells was examined by using SIRC rabbit corneal epithelial cells as model. [Ca2 +]i and cell viability were measured by using the fluorescent dyes fura-2 and WST-1, respectively. Econazole at concentrations ≥ 1 μ M increased [Ca2 +]i in a concentration-dependent manner. The Ca2 + signal was reduced partly by removing extracellular Ca2 +. The econazole-induced Ca2 + influx was insensitive to L-type Ca2 + channel blockers and protein kinase C modulators. In Ca2 +-free medium, after pretreatment with 20 μ M econazole, [Ca2 +]i rises induced by 1 μ M thapsigargin (an endoplasmic reticulum Ca2 + pump inhibitor) were abolished. Conversely, thapsigargin pretreatment also abolished econazole-induced [Ca2 +]i rises. Inhibition of phospholipase C with 2 μ M U73122 did not change econazole-induced [Ca2 +]i rises. At concentrations between 10 and 80 μ M, econazole killed cells in a concentration-dependent manner. The cytotoxic effect of 20 μ M econazole was not reversed by prechelating cytosolic Ca2 + with BAPTA. This shows that in SIRC cells econazole induces [Ca2 +]i rises by causing Ca2 + release from the endoplasmic reticulum and Ca2 + influx from unknown pathways. Econazole-caused cytotoxicity was independent from a preceding [Ca2 +]i rise. [ABSTRACT FROM AUTHOR]

Abstract: Bacillus cereus continues to be one of the important foodborne pathogens due to its ability to produce various heat-labile and -stable toxins. Several methods have been developed to assess the pathogenicity of the B. cereus strains; however, most of these take more than 2–3 days to provide confirmatory results. In this study we standardized a one-step cytotoxicity assay using WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) and compared with the traditional MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide)-based assay for rapid detection of cytotoxic Bacillus spp. using Chinese hamster ovary (CHO) cell line. Crude toxin preparations from 50 isolates of Bacillus spp. were exposed to CHO cell line for 1 h or 24 h and the cytotoxicity was determined by using WST-1 and MTT-based methods. Most B. cereus strains and some strains of other Bacillus species from our collection or from food sources showed comparably high cytotoxicity using either of the methods (P =0.81); however, WST-1 assay provided results in only 3 h while MTT assay in 44–52 h. A positive correlation (R 2 =0.93) between WST-1 and MTT assays strongly suggests that the WST-1-based cytotoxicity assay could be used as an alternative method to MTT assay for rapid (3 h) confirmation of toxigenic Bacillus species in foods prior to their retail distribution or consumption. [Copyright &y& Elsevier]

The effect of the synthetic estrogen diethylstilbestrol (DES) on cytosolic free Ca2+ concentrations ([Ca2+]i) and cell viability was explored in Chinese hamster ovary (CHO-K1). [Ca2+]i and cell viability were measured by using the fluorescent dyes fura-2 and WST-1, respectively. DES at concentrations ≥ 1∝ increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca2+. In Ca2+-free medium, after pretreatment with 50∝ DES, 1∝ thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor)-induced [Ca2+]i rises were abolished. Conversely, thapsigargin pretreatment abolished DES-induced [Ca2+]i rises. Inhibition of phospholipase C with U73122 did not alter DES-induced [Ca2+]i rises. At a concentration of 5∝, DES increased cell viability. At concentrations of 100-200 μ M, DES decreased viability in a concentration-dependent manner. The effect of 5 and 100 μM DES on viability was partly reversed by prechelating cytosolic Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N' -tetraacetic acid (BAPTA). DES-induced cell death was induced via apoptosis as demonstrated by propidium iodide staining. DES (100 μ M)-induced [Ca2+]i rises were largely inhibited by pretreatment with the estrogen receptor antagonist ICI-182,780 (100 μ M). ICI-182,780 did not affect 5 μ M DES-induced increase in viability but partly reversed 100 μ M DES-induced cell death. Collectively, in CHO-K1 cells, DES induced [Ca2+]i rises by stimulating estrogen receptors leading to Ca2+ release from the endoplasmic reticulum in a phospholipase C-independent manner, and Ca2+ influx. DES-caused cytotoxicity was mediated by an estrogen receptor- and Ca2+-dependent pathway. [ABSTRACT FROM AUTHOR]

Abstract: Background: Hb interference induces false test results and thus hinders the establishment of further applications that use the formazan method. We attempted to eliminate hemoglobin (Hb) interference on the formazan reaction. Methods: Total serum bile acid (TBA) reagent was constructed to elucidate the mechanism of Hb interference. We determined the interaction between formazan reagents and Hb using spectral analysis and investigated the optimal condition of the reagent to establish a practical technique for its prevention. Results: Hb caused overestimation during TBA measurement, and its influence was attributed to the errors of both the sample and reagent blank signals. These errors were triggered by the reaction between Hb and formazan reagents. The main finding was that the addition of both imidazole and sodium nitrite in the reagent considerably accelerated the oxidation of Hb, even at neutral pH. The overestimation of TBA concentration was reduced to <3 μmol/l even with Hb concentrations ≥500 mg/dl. Conclusions: We elucidated the mechanism of Hb interference on the formazan reaction and succeeded in preventing its influence. This will help to solve the technical difficulties associated with utilizing formazan reactions as routine diagnostic tools. [Copyright &y& Elsevier]