The need to compare: assessing the level of agreement of three high-throughput assays against Plasmodium falciparum mature gametocytes.

Whole-cell High-Throughput Screening (HTS) is a key tool for the discovery of much needed malaria transmission blocking drugs. Discrepancies in the reported outcomes from various HTS Plasmodium falciparum gametocytocidal assays hinder the direct comparison of data and ultimately the interpretation of the transmission blocking potential of hits. To dissect the underlying determinants of such discrepancies and assess the impact that assay-specific factors have on transmission-blocking predictivity, a 39-compound subset from the Medicines for Malaria Venture Malaria Box was tested in parallel against three distinct mature stage gametocytocidal assays, under strictly controlled parasitological, chemical, temporal and analytical conditions resembling the standard membrane feeding assay (SMFA). Apart from a few assay-specific outliers, which highlighted the value of utilizing multiple complementary approaches, good agreement was observed (average ΔpIC50 of 0.12 ± 0.01). Longer compound incubation times improved the ability of the least sensitive assay to detect actives by 2-fold. Finally, combining the number of actives identified by any single assay with those obtained at longer incubation times yielded greatly improved outcomes and agreement with SMFA. Screening compounds using extended incubation times and using multiple in vitro assay technologies are valid approaches for the efficient identification of biologically relevant malaria transmission blocking hits.

Anticancer activity of biologically synthesized silver and gold nanoparticles on mouse myoblast cancer cells and their toxicity against embryonic zebrafish.

The aim of this study was to evaluate the anticancer activity of bioinspired silver nanoparticles (AgNPs) and gold nanoparticles (AuNPs) against mouse myoblast cancer cells (C2C12). Both AgNPs and AuNPs were biologically synthesized using Spinacia oleracea Linn., aqueous leaves extract. UV-Vis. spectrophotometer, high resolution-transmission electron microscopy (HR-TEM), field emission-scanning electron microscopy (FE-SEM) and X-ray diffraction (XRD) studies supported the successful synthesis of AgNPs and AuNPs. Both these NPs have shown cytotoxicity against C2C12 cells even at very low concentration (5µg/mL). Acridine orange/Ethidium bromide (AO/EB) dual staining confirmed the apoptotic morphological features. The levels of caspase enzymes (caspase-3 and caspase-7) were significantly up-regulated in NPs treated myoblast cells than the plant extract. Furthermore, in zebrafish embryo toxicity study, AgNPs showed 100% mortality at 3µg/mL concentration while AuNPs exhibited the same at much higher concentration (300mg/mL). Taken together, these results provide a preliminary guidance for the development of biomaterials based drugs to fight against the fatal diseases for example cancer.

Antigenotoxic and apoptotic activity of green tea polyphenol extracts on hexavalent chromium-induced DNA damage in peripheral blood of CD-1 mice: analysis with differential acridine orange/ethidium bromide staining.

This study was conducted to investigate the modulating effects of green tea polyphenols on genotoxic damage and apoptotic activity induced by hexavalent chromium [Cr (VI)] in CD-1 mice. Animals were divided into the following groups: (i) injected with vehicle; (ii) treated with green tea polyphenols (30 mg/kg) via gavage; (iii) injected with CrO3 (20 mg/kg) intraperitoneally; (iv) treated with green tea polyphenols in addition to CrO3. Genotoxic damage was evaluated by examining micronucleated polychromatic erythrocytes (MN-PCEs) obtained from peripheral blood at 0, 24, 48, and 72 h after treatment. Induction of apoptosis and cell viability were assessed by differential acridine orange/ethidium bromide (AO/EB) staining. Treatment of green tea polyphenols led to no significant changes in the MN-PCEs. However, CrO3 treatment significantly increased MN-PCEs at 24 and 48 h after injection. Green tea polyphenols treatment prior to CrO3 injection led to a decrease in MN-PCEs compared to the group treated with CrO3 only. The average of apoptotic cells was increased at 48 h after treatment compared to control mice, suggesting that apoptosis could contribute to eliminate the DNA damaged cells induced by Cr (VI). Our findings support the proposed protective effects of green tea polyphenols against the genotoxic damage induced by Cr (VI).

Improved efficacy of fluconazole against candidiasis using bio-based microemulsion technique.

Candida albicans is a common fungal pathogen that causes systemic and superficial infections in most immunocompromised patients. Fluconazole, a synthetic triazole antifungal agent, is the most prescribed drug used in treating this pathogen. But because of its poor solubilization in water and the emergence of resistant strains against this antimycotic drug, we aimed at devising a unique microemulsion drug delivery system for fluconazole against candidiasis. A clear oil-in-water microemulsion system, consisting of clove oil as oil phase, Tween 20 as surfactant, and water as aqueous phase was developed using a ternary phase diagram. Physicochemical characterization was done to understand the internal physicochemical state. The bulk drug, fluconazole, that measured several microns in length was reduced to a 10-65 nm range with no means of high-energy methods as confirmed by transmission electron microscopy. The very small and uniform spherical structure of the drug-loaded microemulsion system could be of high impact to the biological system as the efficacy of fluconazole is greatly improved when compared with its conventional bulk form. The optimized microemulsion exhibited significantly higher antifungal activity at a minimum concentration (8 µg/ml) of fluconazole as examined by fluorescence and scanning electron microscopy. Thus, our report discloses an excellent oral drug delivery system.

Effects of ascorbic acid and ß-carotene on HepG2 human hepatocellular carcinoma cell line.

Recent studies have demonstrated that vegetable rich diets have protective effects on the occurrence and prognosis of various cancers. In addition to dietary intakes, ascorbic acid and ß-carotene are also taken as supplements. The aim of this study was to assess effects of ascorbic acid, ß-carotene and their combinations on human hepatocellular carcinoma cell line HepG2. Ascorbic acid and ß-carotene were applied to cells as plasma peak concentrations (70 and 8 µM, respectively) and their half concentrations (35 and 4 µM, respectively) for 24 and 48 h. Genotoxic and cytotoxic effects of ascorbic acid and ß-carotene were evaluated by alkali single cell gel electrophoresis (SCGE), acridine orange/ethidium bromide staining patterns of cells (apoptosis and necrosis) and lipid peroxidation (thiobarbituric acid reactive substances, TBARS). Results of the SCGE demonstrated that both ascorbic acid and ß-carotene caused DNA damage on HepG2 which were also concordant to increased apoptosis and necrosis of cells. Increased TBARS values also demonstrated increased lipid peroxidation in these cells. Results of the present study demonstrates that when dietary intakes of ascorbic acid and ß-carotene and their relevant achievable plasma level concentrations were considered, both ascorbic acid and ß-carotene induce genotoxic and cytotoxic damage on HepG2 together with increased oxidative damage in contrast to their protective effect on healthy cells. This may be correlated to oxidative status and balance of ROS in hepatocellular carcinoma cells.

Kauren-19-oic acid induces DNA damage followed by apoptosis in human leukemia cells.

This study evaluated the potential cytotoxicity of the natural diterpenoids kauren-19-oic acid (KA), 14-hydroxy-kaurane (1) and xylopic acid (2), and semi-synthetic derivatives of KA (3-5) towards human cancer cell lines (K562, HL60, MDA-MB435 and SF295) and lymphocytes. Mouse erythrocytes were used to verify a possible hemolytic activity Cytotoxicity mechanisms were investigated in HL60 cells. KA showed a moderate antiproliferative effect in MTT assay towards all cancer cells (IC(50), 9.1-14.3 microg ml(-1)). However, KA appeared not selective to cancer cells, since it also inhibited the lymphocytes proliferation (IC(50), 12.6 microg ml(-1)). Unlike KA, compounds 1-5 displayed no cytotoxicity and were also free from antiproliferative and hemolytic effects, suggesting that the exocyclic double bond (C16) unit may be the active pharmacophore of KA cytotoxicity. KA-treated HL60 cells displayed decreased proliferation (5-bromo-2';-deoxyuridine incorporation assay) and topoisomerase I activity (DNA relaxation assay). These assays revealed that KA primarily intercalates with DNA and not with topoisomerase I. Fluorescence microscopy using AO/EB (acridine orange/ethidium bromide) staining indicated that KA can induce both apoptosis and necrosis in HL-60 cell cultures, which corroborate the findings with MTT. From these findings, we conclude that KA, although demonstrating cytotoxic potential, may have a limited or poor therapeutic potential due to lack of selectivity to tumor cells. Further studies on the structure modification of KA and the mechanism of the new derivatives are currently in progress.

Antimutagenic in vitro activity of plant polyphenols: Pycnogenol and Ginkgo biloba extract (EGb 761).

Ofloxacin (15 microg/mL) and acridine orange (5 microg/mL) induce mutagenicity by different mechanisms in the photosynthetic flagellate Euglena gracilis. The present study examined whether Pycnogenol (PYC; 5-100 microg/mL) or Ginkgo biloba extract (EGb 761; 5-100 microg/mL) could protect against the mutagenic effects of each of the mutagens and the potential mechanisms underlying such protection. The highest concentration of PYC and EGb 761 effectively reduced the mutagenic activity of both ofloxacin and acridine orange by more than 99% (p < 0.001). Using luminol-dependent photochemical methodology it was demonstrated that EGb 761 and PYC were effective antioxidants. In addition, as determined by spectrophotometry, PYC and EGb 761 bound acridine orange. Both PYC and EGb 761 have been shown to produce dual antimutagenic effects, as evidenced by both antioxidant and physicochemical properties. The findings suggest that EGb 761 and PYC would thus be suitable for future study, not only as antioxidants, but also as antimutagenic agents.