Exploration of novel in vitro assays to study drugs against Trichuris spp

Though trichuriasis is a significant public health problem, few effective drugs are available underscoring the need for new drug therapies. For the evaluation of trichuricidal activity of test compounds in vitro an accurate, reliable, sensitive, fast and cheap drug sensitivity assay is essential. The aim of the present investigation was to evaluate the performance of different in vitro drug sensitivity assays in comparison to the standard motility assay. Trichuris muris L4 larvae or adult worms were isolated from the intestinal tract from infected female C57BL/10 mice and incubated in the presence of ivermectin, levamisole and nitazoxanide (200, 100 and 50 μg/ml) for 72 h. The health status of the worms was either evaluated microscopically using a motility scale from 0 to 3 (motility assay), by examination of absorbance or emission in response to metabolic activity (MTT (Thiazolyl Blue Tetrazolium Bromide) and Alamar Blue assay), through analysis of absorbance of an enzyme-substrate reaction (acid phosphatase activity assay), by measuring the noise amplitudes (isothermal microcalorimetry and xCELLigence System) or the heat flow (isothermal microcalorimetry) of T. muris. The Alamar Blue assay, xCELLigence and microcalorimetry compared favorably to the standard motility assay. These three assays precisely determined the trichuricidal activity of the three test drugs. The acid phosphatase and the MTT assays showed a poorer performance than the motility assay. In conclusion, the colorimetric Alamar Blue in vitro assay is a good alternative to the motility assay to study drug effects against T. muris L4 and adults, since it is easy to perform, precise and of low cost.     

Bacteriological follow-up of pulmonary tuberculosis treatment: a study with a simple colorimetric assay

The viability of Mycobacterium tuberculosis (MTB) in serial sputum specimens from persistently smear positive patients was evaluated. The assay was based on oxidation-reduction of Alamar Blue and Malachite Green dyes that change their color in response to MTB growth. A total of 280 sputum specimens from 40 persistently smear positive TB patients and 40 sputa from non-tuberculosis patients were digested, decontaminated and examined microscopically. To check the MTB viability, the sediments from decontaminated samples were inoculated into three culture media: Lowenstein-Jensen (LJ) slants, Alamar Blue and Malachite Green culture tubes. We found that out of 280 smear positive specimens, the LJ culture was positive in 124 (44%). The numbers of correctly identified S+/C+ cases by Alamar Blue and Malachite Green were 118 (95%) and 116 (93%), respectively. The mean time required for reporting the positive signal in Alamar Blue culture tubes was 9 versus 11 days by Malachite Green culture tubes. In the standard LJ culture media the average detection time was 27 days (P < 0.05). The sensitivity of LJ was 99%, Alamar Blue 95% and Malachite Green 93%. The specificity was 100%, 92% and 93%, respectively. The oxidation-reduction method is rapid, sensitive and inexpensive in monitoring the treatment response of patients with pulmonary TB. Thus, using this method can be of paramount importance, particularly in resource-constrained areas.     

The photo comet assay--a fast screening assay for the determination of photogenotoxicity in vitro

Different classes of chemicals can induce a phototoxic effect by absorbing light energy within the wavelength range of sunlight. The assessment of photo-safety is therefore an obligatory part of the development of new drugs. Ten UV-vis (280-800nm)-absorbing compounds (ketoprofen, promazine, chlorpromazine, dacarbazine, acridine, lomefloxacin, 8-methoxypsoralen, chlorhexidine, titanium dioxide, octylmethoxycinnamate) were tested for their photogenotoxic potential in the alkaline comet assay in the presence and absence of UV-vis. In order to establish an easy and timesaving protocol for a photo comet assay screening test, the application of 96-well plates was essential. The use of mouse lymphoma L5178Y cells, a cell line growing in suspension, allowed the determination of photocytotoxicity with the Alamar Blue assay and of photogenotoxicity with the alkaline comet assay in parallel. L5178Y cells were incubated with the test compounds for 20min and irradiated with simulated sunlight in the wavelength range from 280 to 800nm. The applied UV dose was 600mJ/cm(2) UV-A and 30mJ/cm(2) UV-B. After a post-incubation of 10min, the Alamar Blue assay and the alkaline comet assay were performed. All of the compounds which are known to be photogenotoxic (8-methoxypsoralen, acridine, chlorpromazine, dacarbazine, ketoprofen, lomefloxacin) showed a positive effect under our assay conditions. Furthermore, four UV-vis absorbing chemicals which are known to be not photogenotoxic (promazine, chlorhexidine, titanium dioxide, octylmethoxycinnamate) were analysed. For none of them an increase of the DNA damage following irradiation was observed in this study. In conclusion, all of the chemical compounds tested were classified in agreement with published data. From the data presented it is concluded that the photo comet assay with L5178Y mouse lymphoma cells is a reliable model to assess photochemical genotoxicity in vitro.     

Synthesis and in vitro anticancer and antitubercular activity of diarylpyrazole ligated dihydropyrimidines possessing lipophilic carbamoyl group

A series of dihydropyrimidine derivatives were synthesized by utilizing Biginelli reaction and evaluated for their in vitro anticancer activity against MCF-7 human breast cancer (HBC) cell line using sulforhodamine B (SRB) assay and antitubercular activity against Mycobacterium tuberculosis (MTB) H(37)Rv using Microplate Alamar Blue Assay (MABA). Compounds 13p, 13t were exhibited 70.6% and 63.7% of HBC cell growth inhibition at 10 μM concentration. Interestingly compound 13p was also found to be the most potent in the series against MTB H(37)Rv with MIC value of 0.125 μg/mL.     

Propidium iodide-based methods for monitoring drug action in the kinetoplastidae: comparison with the Alamar Blue assay

The urgent need for new drug development for African trypanosomiasis is widely recognized. This requires reliable and informative high-throughput assays. Currently, drug action is determined with a fluorimetric/colorimetric assay based on the metabolism of the dye Alamar Blue (resazurin) by live cells. However, this assay does not easily distinguish between cell death and growth arrest, or supply information about the rate at which test compounds affect these parameters. We report here an alternative fluorimetric assay, based on the interaction of propidium iodide with DNA, that allows either real-time monitoring of cell viability or the generation of EC₅₀ values at a predetermined time-point. The assay is highly sensitive and fluorescence readings easily correlate to numbers of parasites or DNA content. The EC₅₀ values were highly similar to those obtained with the standard Alamar Blue assay. The procedure lends itself readily to applications in drug development or resistance monitoring.     

Synthesis and anti-tuberculosis activity of glycitylamines

A series of glycitylamines, which were prepared in few steps from readily available carbohydrates, were tested for their ability to inhibit tuberculosis growth in an Alamar Blue BCG colourimetric assay. Several derivatives, including (2R,3R)-1-(hexadecylamine)pent-4-ene-2,3-diol, (2R,3R)-1-(hexadecylmethylamino)pent-4-ene-2,3-diol and 5-deoxy-5-hexadecylmethylamino-d-arabinitol, were prepared in good to excellent (44–90%) overall yield and exhibited micromolar (20–41 μM) inhibitory activity that was similar to the first line tuberculosis (TB) drug ethambutol (39 μM) in the same assay. The ease and low cost of the synthesis of the glycitylamines offers definite advantages for their use as potential TB drugs.     

Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity.

We show here the identity of Alamar Blue as resazurin. The 'resazurin reduction test' has been used for about 50 years to monitor bacterial and yeast contamination of milk, and also for assessing semen quality. Resazurin (blue and nonfluorescent) is reduced to resorufin (pink and highly fluorescent) which is further reduced to hydroresorufin (uncoloured and nonfluorescent). It is still not known how this reduction occurs, intracellularly via enzyme activity or in the medium as a chemical reaction, although the reduced fluorescent form of Alamar Blue was found in the cytoplasm and of living cells nucleus of dead cells. Recently, the dye has gained popularity as a very simple and versatile way of measuring cell proliferation and cytotoxicity. This dye presents numerous advantages over other cytotoxicity or proliferation tests but we observed several drawbacks to the routine use of Alamar Blue. Tests with several toxicants in different cell lines and rat primary hepatocytes have shown accumulation of the fluorescent product of Alamar Blue in the medium which could lead to an overestimation of cell population. Also, the extensive reduction of Alamar Blue by metabolically active cells led to a final nonfluorescent product, and hence an underestimation of cellular activity.     

GTSF-2: A new, versatile cell culture medium for diverse normal and transformed mammalian cells

Summary The aim of this study was to test the versatility of a new basal cell culture medium, GTSF-2. In addition to traditional growth-factors, GTSF-2 contains a blend of three sugars (glucose, galactose, and fructose) at their physiological levels. For these studies, we isolated normal endothelial cells from human, bovine, and rat large blood vessels and microvessels. In addition, GTSF-2 was also tested as a replacement for high-glucose-containing medium for PC12 pheochromocytoma cells and for other, transformed cell lines. The cell growth characteristics were assessed with a novel cell viability and proliferation assay, which is based on the bioreduction of the fluorescent dye, Alamar Blue. After appropriate calibration, the Alamar Blue assay was found to be equivalent to established cell proliferation assays. Alamar Blue offers the advantage that cell proliferation can be measured in the same wells over an extended period of time. For some of the cell types (e.g., endothelial cells isolated from the bovine aorta, the rat adrenal medulla, or the transformed cells), proliferation in unmodified GTSF-2 was equivalent to that in the original culture media. For others cell types (e.g., human umbilical vein endothelial cells and PC12 cells), GTSF-2 proved to be a superior growth medium, when supplemented with simple additives, such as endothelial cell growth supplement (bFGF) or horse serum. Our results suggest that GTSF-2 is a versatile basal medium that will be useful for studying organ-specific differentiation in heterotypic coculture studies.     

The reduction of Alamar Blue by peripheral blood lymphocytes and isolated mitochondria

Alamar Blue is a widely used nontoxic indicator of cell proliferative activity, which penetrates quickly through the biological membranes and can be easily reduced by intracellular enzymes. Accumulation of reduced fluorescent form of Alamar Blue during short-term culture of human peripheral blood lymphocytes may be used as a cell viability test since it was prevented by disruption of plasma membrane by digitonin. The inhibition of Alamar Blue reduction by NaN3 indicates that its metabolism is associated with mitochondrial activity. A compaative study of Alamar Blue reduction and oxygen consumption on isolated rat liver mitochondria shows, that the Alamar Blue reduction is not associated with the activity of specific complex of respiratory chain and it seems to be an integral indicator of oxidation-reduction activity of respiratory chain components.     

Study of hyaluronan synthase inhibitor, 4-methylumbelliferone derivatives on human pancreatic cancer cell (KP1-NL)

The structure of 4-methylumbelliferone (MU) consists of coumarin with 4-methyl group and 7-hydroxy group. MU inhibits HA synthesis and pericellular HA matrix formation. In this study, we used 10 MU derivatives which have hydroxy groups and methyl groups at various positions of coumarin to investigate a more effective HA inhibitor than MU. First, human pancreatic cancer cell (KP1-NL) growth assay was analyzed by Alamar Blue to determine the non-toxic concentration of MU derivatives, and the inhibitory effect on HA synthesis in the cell cultures was analyzed by HA measuring kit. Next, cell surfaces of cancer cells were analyzed by particle-exclusion assay. In conclusion, both hydroxy and methyl groups are necessary for HA inhibition by MU, and two hydroxy groups inhibited HA synthesis more strongly than MU.     

Synthesis and antituberculosis activity of new 3-alkylsulfanyl-1,2,4-triazole derivatives

The increasing clinical importance of drug-resistant mycobacterial pathogens has lent additional urgency to microbiological research and new antimycobacterial compound development. For this purpose, new alkylsulfanyltriazoles were synthesized and evaluated for antituberculosis activity. The reaction of thienyl-2-acetic acid with thiocarbohydrazide gave the mercaptotriazoles (II). The 4-amino-5-(2-thienylmethyl)-3-[1-(2-thienyl)-3-aryl) propion-3-yl] sulfanyl-4H-1,2,4-triazole (III) derivatives were synthesized by reacting the mercaptotriazoles with chalcones (I). Antituberculosis activities of the synthesized compounds were determined by broth microdilution assay, the Microplate Alamar Blue Assay, in BACTEC 12B medium and results were screened in-vitro, using BACTEC 460 Radiometric System against Mycobacterium tuberculosis H37Rv (ATCC 27294) at 6.25 microg/ml and the tested compounds showed considerable inhibition ranging from 58-84%.     

C-Dimethylated Flavones as Possible Potential Anti-Tubercular and Anticancer Agents.

The present investigation describes an intramolecular Oxa-Michael addition of penta-substituted phenols to the enone of the tether in the presence of iodine as the oxidizing agent. Ten C-Dimethylated flavones with moderate to good yields ( 10a – j , 60–89 %) were isolated by heating the corresponding C-dimethylated chalcones using iodine in DMSO. Using the Microplate Alamar Blue test (MABA) technique, the drugs′ quantitative drug susceptibility against the H37Rv strain of replicating Mycobacterium TB was determined. The sensitivity of two of the developed compounds ( 10e , 10h ) was up to 6.25 g/mL. The human lung adenocarcinoma cell lines (A549) were used in the anticancer study, which was carried out using the MTT cell proliferation assay. In A549 cell lines, four flavones demonstrated anticancer activity with IC₅₀ values between 39 and 48 μM. The C-dimethylated flavones, 10b (3,4-dimethoxy), 10c (2,3,4-trimethoxy), 10e (p-fluoro) and 10 g (N-methyl indole) substitutions on ring ‘B’ showed good anticancer activity with IC₅₀ values 39.17, 39.21, 48.43 and 43.48 μM, respectively. The compounds 10b , 10c , 10d , 10e , and 10i had improved binding and interaction profiles among all the compounds examined during the current In Silico research, as shown by the docking simulations against two targets EGFR and MTB MurI.     

Establishment and characterization of permanent cell line from gill tissue of Labeo rohita (Hamilton) and its application in gene expression and toxicology

Rohu gill cell line (LRG) was established from gill tissue of Indian major carp (Labeo rohita), a freshwater fish cultivated in India. The cell line was maintained in Leibovitz's L-15 supplemented with 10 % foetal bovine serum (FBS). This cell line has been sub-cultured more than 85 passages over a period of 2 years. The LRG cell line consists of both epithelial and fibroblastic-like cells. The cells were able to grow at a wide range of temperatures from 22 to 32 °C, the optimum temperature being 28 °C. The growth rate of gill cells increased as the FBS proportion increased from 2 to 20 % at 28 °C. The plating efficiency was also high (34.37 %). The viability of the LRG cell line was 70–80 % after 6 months of storage in liquid nitrogen. The karyotype analysis revealed a diploid count of 50 chromosomes. The gill cells of rohu were successfully transfected with pEGFP-N1. Amplification of mitochondrial Cox1 gene using primers specific to L. rohita confirmed the origin of this cell line from L. rohita. The cytotoxicity of malathion was assessed in LRG cell line using multiple endpoints such as 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Neutral Red assay, Alamar Blue assay and Coomassie Blue protein assay. Acute toxicity assay on fish was conducted by exposing L. rohita for 96 h to malathion under static conditions. Statistical analysis revealed good correlation with r ²?=?0.946–0.990 for all combinations between endpoints employed. Linear correlations between each in vitro effective concentration 50 and the in vivo lethal concentration 50 data were highly significant.     

A high density assay format for the detection of novel cytotoxic agents in large chemical libraries

In response to the need for inexpensive high throughput assays for anti-cancer drug screening, a 1536-well microtiter plate based assay utilizing the Alamar Blue fluorescent dye as a measure of cellular growth was validated in 10 μL assay volume. Its robustness was assessed in a screen against a library of 2000 known bioactives; with an overall Z′ value of 0.89 for assay robustness, several known cytotoxic agents were identified including and not limited to anthracyclines, cardiac glycosides, gamboges, and quinones. To further test the sensitivity of the assay, IC₅₀ determinations were performed in both 384-well and 1536-well formats and the obtained results show a very good correlation between the two density formats. These findings demonstrate that this newly developed assay is simple to set up, robust, highly sensitive and inexpensive. It could potentially provide a rapid way to screen established and primary tumor cell lines against large chemical libraries.     

A high density assay format for the detection of novel cytotoxic agents in large chemical libraries

In response to the need for inexpensive high throughput assays for anti-cancer drug screening, a 1536-well microtiter plate based assay utilizing the Alamar Blue fluorescent dye as a measure of cellular growth was validated in 10 microL assay volume. Its robustness was assessed in a screen against a library of 2000 known bioactives; with an overall Z' value of 0.89 for assay robustness, several known cytotoxic agents were identified including and not limited to anthracyclines, cardiac glycosides, gamboges, and quinones. To further test the sensitivity of the assay, IC50 determinations were performed in both 384-well and 1536-well formats and the obtained results show a very good correlation between the two density formats. These findings demonstrate that this newly developed assay is simple to set up, robust, highly sensitive and inexpensive. It could potentially provide a rapid way to screen established and primary tumor cell lines against large chemical libraries.     

Determination of interleukin 2 activity by a new fluorometric method

Summary Interleukin 2 activity is usually determined by a proliferation assay using an IL-2-dependent cell line. Tritiated thymidine incorporation during DNA synthesis is a suitable method for this purpose, but its main drawback is the use of radioactive isotopes. We describe the use of Alamar Blue, a new fluorogenic growth indicator, for the measurement of interleukin 2 activity in microtitration plates. This assay is sensitive and economical. The lower limit of detection is about 400 cells per well with an intra-assay coefficient of variation of about 5 percent.     

Evaluation of a porcine lens and fluorescence assay approach for in vitro ocular toxicological investigations

Cell biology, as monitored with the fluorescent indicator dyes Alamar Blue and 5-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM), and lens optical quality, as measured with an in vitro scanning laser system, have been used to evaluate in vitro the condition of porcine lenses after being placed in a culture medium. The measurements, beginning from week one of culture, were compared statistically. Optical quality and cellular viability, as measured with either dye, were unchanged in lenses that had been maintained for 6 weeks in modified M199 medium. Some lenses were treated with 0.152J/cm(2) UVB radiation, and a decline was observed after 48 hours in both optical and metabolic capabilities, as indicated by a decreased capacity of the lenses to reduce Alamar Blue. The measurements with CFDA-AM did not show complete concordance with the other indicators of lens health after UV treatment, making this dye less reliable as applied currently to lens cultures. Overall, the findings suggest that porcine lenses can be maintained for weeks in culture, and that their condition can be evaluated quantitatively by assays that probe cellular functions and optical properties. Such a system should prove valuable for in vitro ocular pharmacotoxicological research.     

Synthesis and antituberculosis activity of new 3-alkylsulfanyl-1,2,4-triazole derivatives

The increasing clinical importance of drug-resistant mycobacterial pathogens has lent additional urgency to microbiological research and new antimycobacterial compound development. For this purpose, new alkylsulfanyltriazoles were synthesized and evaluated for antituberculosis activity. The reaction of thienyl-2-acetic acid with thiocarbohydrazide gave the mercaptotriazoles (II). The 4-amino-5-(2-thienylmethyl)-3-[1-(2-thienyl)-3-aryl)propion-3-yl]sulfanyl-4H-1,2,4-triazole (III) derivatives were synthesized by reacting the mercaptotriazoles with chalcones (I). Antituberculosis activities of the synthesized compounds were determined by broth microdilution assay, the Microplate Alamar Blue Assay, in BACTEC12B medium and results were screened in-vitro, using BACTEC 460 Radiometric System against Mycobacterium tuberculosis H₃₇Rv (ATCC 27294) at 6.25?μg/ml and the tested compounds showed considerable inhibition ranging from 58–84%.     

Specific and dynamic detection of palytoxins by in vitro microplate assay with human neuroblastoma cells

Palytoxin is one of the most complex and biggest molecules known to show extreme acute toxicity. The dinoflagellate Ostreopsis spp., the producer organism of palytoxin, has been shown to be distributed worldwide, thus making palytoxin an emerging toxin. Rat-derived hepatocytes (Clone 9) and BE (2)-M17 human neuroblastoma cells were used to test palytoxin or palytoxin-like compounds by measuring the cell metabolic rate with Alamar Blue. The dose-dependent decrease in viability was specifically inhibited by ouabain in the case of BE (2)-M17 neuroblastoma cells. This is a functional, dynamic and simple test for palytoxins with high sensitivity (as low as 0.2 ng/ml). This method was useful for toxin detection in Ostreopsis extracts and naturally contaminated mussel samples. A comparative study testing toxic mussel extracts by LC (liquid chromatography)-MS/MS (tandem MS), MBA (mouse bioassay), haemolysis neutralization assay and a cytotoxicity test indicated that our method is suitable for the routine determination and monitoring of palytoxins and palytoxin-like compounds.     

Synthesis and in vitro antitubercular activity of a series of quinoline derivatives

A series of 33 quinoline derivatives have been synthesized and evaluated for their in vitro antibacterial activity against Mycobacterium tuberculosis H₃₇Rv using the Alamar Blue susceptibility test and the activity expressed as the minimum inhibitory concentration (MIC) in μg/mL. Compounds 5e and 5f exhibited a significant activity at 6.25 and 3.12 μg/mL, respectively, when compared with first line drugs such as ethambutol and could be a good starting point to develop new lead compounds in the fight against multi-drug resistant tuberculosis.