Alamar BlueTM Research Articles

3D scaffolds of caprolactone/chitosan/polyvinyl alcohol/hydroxyapatite stabilized by physical bonds seeded with swine dental pulp stem cell for bone tissue engineering

Bone Regeneration represents a clinical need, related to bone defects such as congenital anomalies, trauma with bone loss, and/or some pathologies such as cysts or tumors This is why a polymeric biomaterial that mimics the osteogenic composition and structure represents a high potential to face this problem. The method of obtaining these materials was first to prepare a stabilized hydrogel by means of physical bonds and then to make use of the lyophilization technique to obtain the 3D porous scaffolds with temperature conditions of −58 °C and pressure of 1 Pa for 16 h. The physicochemical and bioactive properties of the scaffolds were studied. FTIR and TGA results confirm the presence of the initial components in the 3d matrix of the scaffold. The scaffolds exhibited a morphology with pore size and interconnectivity that promote good cell viability. Together, the cell viability and proliferation test, Alamar BlueTM and the differentiation test: alizarin staining, showed the ability of physically stabilized scaffolds to proliferate and differentiate swine dental pulp stem cell (DPSCs) followed by mineralization. Therefore, the Cs-PCL-PVA-HA scaffold stabilized by physical bonds has characteristics that suggest great utility for future complementary in vitro tests and in vivo studies on bone defects. Likewise, this biomaterial was enhanced with the addition of HA, providing a scaffold with osteoconductive properties necessary for good regeneration of bone tissue.Graphical abstract

Comparison of the Biological Behavior and Topographical Surface Assessment of a Minimally Invasive Dental Implant and a Standard Implant: An In Vitro Study.

The current study aimed to assess the topographical and physical properties of a minimally invasive implant (MagiCore®: MC®, InnosBioSurg, IBS) and to evaluate its biological behavior compared to a gold standard implant (NobelParallel™: NB™, Nobel Biocare™). After surface characterization, the biological behavior assessment was conducted regarding human gingival fibroblasts (hGF) and osteoblast-like cells (MG63). Roughness values for NBTM were Ra = 1.28 µm and for MC® they were Ra = 2.02 µm. Alamar BlueTM assay LIVE/DEADTM staining results indicated equivalent biological development regarding both cell types for the two implants. Significant enhancement was found for hGF ALP activity in the presence of the two tested implants in a time-dependent manner from day 7 to day 14 (** p < 0.01). Alizarin red staining demonstrated significant calcium deposition enhancement when cells were interfaced with the NB™ compared to the MC® implant (** p < 0.05). Moreover, SEM and confocal imaging revealed good cell adhesion with a denser cellular layer on the MC® than the NB™ surface. The MC® cytocompatibility was ranked as equivalent to the gold standard implant despite the surface properties differences. These findings provide new insights about the minimally invasive implant's biological behavior and its potential clinical implication in different implantology situations.

Developmental neurotoxicity of acrylamide and its metabolite glycidamide in a human mixed culture of neurons and astrocytes undergoing differentiation in concentrations relevant for human exposure

Neural stem cells (NSCs) derived from human induced pluripotent stem cells were used to investigate effects of exposure to the food contaminant acrylamide (AA) and its main metabolite glycidamide (GA) on key neurodevelopmental processes. Diet is an important source of human AA exposure for pregnant women, and AA is known to pass the placenta and the newborn may also be exposed through breast feeding after birth. The NSCs were exposed to AA and GA (1 ×10−8 – 3 ×10−3 M) under 7 days of proliferation and up to 28 days of differentiation towards a mixed culture of neurons and astrocytes. Effects on cell viability was measured using Alamar Blue™ cell viability assay, alterations in gene expression were assessed using real time PCR and RNA sequencing, and protein levels were quantified using immunocytochemistry and high content imaging. Effects of AA and GA on neurodevelopmental processes were evaluated using endpoints linked to common key events identified in the existing developmental neurotoxicity adverse outcome pathways (AOPs). Our results suggest that AA and GA at low concentrations (1 ×10−7 - 1 ×10−8 M) increased cell viability and markers of proliferation both in proliferating NSCs (7 days) and in maturing neurons after 14–28 days of differentiation. IC50 for cell death of AA and GA was 5.2 × 10−3 M and 5.8 × 10−4 M, respectively, showing about ten times higher potency for GA. Increased expression of brain derived neurotrophic factor (BDNF) concomitant with decreased synaptogenesis were observed for GA exposure (10−7 M) only at later differentiation stages, and an increased number of astrocytes (up to 3-fold) at 14 and 21 days of differentiation. Also, AA exposure gave tendency towards decreased differentiation (increased percent Nestin positive cells). After 28 days, neurite branch points and number of neurites per neuron measured by microtubule-associated protein 2 (Map2) staining decreased, while the same neurite features measured by βIII-Tubulin increased, indicating perturbation of neuronal differentiation and maturation.

Direct effect of transforming growth factor-beta 1 (TGF-β1) on human apical papilla cell proliferation and mineralisation.

Firstly, this study investigated the direct effect of Transforming Growth Factor-beta 1 at 10, 5, 2.5 and 1.25 ng mL-1 on human apical papilla cell proliferation and mineralisation. Cell proliferation was examined at 0, 2, 4, 6, 24, 48, 72, 96 and 120 h using Alamar BlueTM assay. Cell mineralisation was examined at day 21 with a quantitative Alizarin Red S staining. Secondly, the study aimed to estimate the amount of Transforming Growth Factor-beta 1 (TGF-β1) released from the dentin after root canal irrigation. The solution collected from a root canal after rinsing with various protocols (normal saline solution, ethylenediaminetetraacetic acid or chlorhexidine) was analysed with an enzyme-linked immunosorbent assay. Data were statistically analysed using a one-way analysis of variance. The results, from the first part, revealed cell proliferation reduction in all experimental groups presented with TGF-β1. The higher concentration generated more deteriorating effects. Cell mineralisation was highest in a group with TGF-β1 at 1.25 ng mL-1 (P < 0.05). For the growth factor released from dentin, the highest amount was detected only when ethylenediaminetetraacetic acid was associated with the irrigation (P < 0.05). In summary, the direct effects of TGF-β1 on cell proliferation and differentiation were diverse, depending on concentration. The release of TGF-β1 from root dentin can be achieved after rinsing with ethylenediaminetetraacetic acid.

Assessment of a PCL-3D Printing-Dental Pulp Stem Cells Triplet for Bone Engineering: An In Vitro Study.

The search of suitable combinations of stem cells, biomaterials and scaffolds manufacturing methods have become a major focus of research for bone engineering. The aim of this study was to test the potential of dental pulp stem cells to attach, proliferate, mineralize and differentiate on 3D printed polycaprolactone (PCL) scaffolds. A 100% pure Mw: 84,500 ± 1000 PCL was selected. 5 × 10 × 5 mm3 parallelepiped scaffolds were designed as a wood-pilled structure composed of 20 layers of 250 μm in height, in a non-alternate order ([0,0,0,90,90,90°]). 3D printing was made at 170 °C. Swine dental pulp stem cells (DPSCs) were extracted from lower lateral incisors of swine and cultivated until the cells reached 80% confluence. The third passage was used for seeding on the scaffolds. Phenotype of cells was determined by flow Cytometry. Live and dead, Alamar blue™, von Kossa and alizarin red staining assays were performed. Scaffolds with 290 + 30 μm strand diameter, 938 ± 80 μm pores in the axial direction and 689 ± 13 μm pores in the lateral direction were manufactured. Together, cell viability tests, von Kossa and Alizarin red staining indicate the ability of the printed scaffolds to support DPSCs attachment, proliferation and enable differentiation followed by mineralization. The selected material-processing technique-cell line (PCL-3D printing-DPSCs) triplet can be though to be used for further modelling and preclinical experiments in bone engineering studies.

P15. Human mesenchymal stem cells exhibit increased proliferation on rough titanium substrates

BACKGROUND CONTEXT In the majority of orthopaedic surgeries, osseointegration between an implant and surrounding bone is crucial to achieving short- and long-term stability of the implant. Since an implant's surface properties directly influence the cellular response at the cell-material interface, a number of techniques exist to modify the surface characteristics of titanium (Ti)-based implants, with the objective of achieving improved osseointegration of the implant and surrounding bone. Many of these processes, such as grit-blasting and acid-etching, create a roughened surface in a random, poorly controlled manner. Conversely, subtractive laser-etching allows for controlled, intentionally designed micro- and nano-scale features to be imparted on a Ti surface. Biomimetic surface modifications can be created utilizing this technique. The biological reactions in response to a roughened, laser-etched Ti-based implant is not well understood. PURPOSE The objective of this study was to evaluate the effect of a roughened, laser-etched Ti6Al4V, substrate designed to mimic the trabecular-like structure of bone, on the cellular behavior of human bone-marrow derived mesenchymal stem cells (BM-MSCs). STUDY DESIGN/SETTING In vitro cell culture study. OUTCOME MEASURES Cell growth and proliferation rate over 7 days of culture were measured using the alamarBlue™ (AB) cell viability kit. At 4 hours, 3 days and 7 days of culture, the redox reaction, in which AB is reduced by the cells, was measured by absorbance readings at 570 and 600 nm. The percent of AB reduction has previously been shown to relate directly to the number of viable cells. METHODS Average 3D surface roughness (Sa) of the TAV substrates was characterized using 3D stereoscopic reconstruction of images from a scanning electron microscope. BM-MSCs were cultured for 7 days in a 24-well plate at a seeding density of 20,000 cells/well on the following substrates: tissue culture polysterene (TCPS), sTAV, rTAV (n=10 per group). Cells were given 4 hours to attach, after which cell media was changed and the TAV substrates were moved to a separate well plate to remove any unattached cells. At 4 hours, 3 days and 7 days of culture, the reduction percentage of AB was measured. The cell proliferation rate was calculated as the rate of change in average AB reduction from the 4 hour to 7 day time points. RESULTS Theaverage roughness of the sTAV and rTAV substrates were 0.36±0.06 μm and 36.3±1.5 μm, respectively. Significantly more AB reduction was found with rTAV substrates compared to sTAV at all time points (p CONCLUSIONS This study found increased cell proliferation on a rough, laser-etched TAV surface designed specifically to mimic the surface morphology of bone when compared to a smooth TAV substrate. This data confirms that surface roughness was increased in a controlled manner, by orders of magnitude, through subtractive laser-etching. It was found that this technique was successful in modifying cellular behavior and resulted in increased BM-MSC proliferation. Future work should investigate how laser-etched rTAV influences cell phenotype, as well as the short- and long-term cellular response. FDA DEVICE/DRUG STATUS Unavailable from authors at time of publication.

Mechanical and surface chemical analysis of retrieved breast implants from a single centre

IntroductionBreast implants are associated with complications such as capsular contracture, implant rupture and leakage often necessitating further corrective surgery. Re-operation rates have been reported to occur in up to 15.4% of primary augmentation patients and up to 27% in primary reconstructions patients within the first three years (Cunningham, 2007). The aim of this study was to examine the mechanical and surface chemical properties as well as the fibroblast response of retrieved breast implants in our unit to determine the in vivo changes which occur over time. MethodsEthical approval was obtained. 47 implants were retrieved. Implantation time ranged from 1 month to 388 months (Mean 106.1 months). Tensile strength, elongation, Young's modulus and tear strength properties were measured using Instron 5565 tensiometer on anterior and posterior aspects of the implant. Attenuated total reflectance-fourier transform infra-red spectroscopy (ATR-FTIR), wettability and scanning electron microscopy (SEM) analysis was performed on the shell surfaces. Bicinchoninic acid assay was performed to determine shell protein content. The fibroblast response was determined by seeding HDFa cells on the retrieved implants and cell metabolism measured using Alamar Blue™ assay. ResultsMechanical properties fall with increasing duration of implantation. There were no significant changes in ATR-FTIR spectra between ruptured and intact implants nor significant changes in wettability in implants grouped into 5 year categories. SEM imaging reveals surface degradation changes with increasing duration of implantation. ConclusionsWith increasing duration of implantation, mechanical properties of the breast implants fall. However this was not associated with surface chemical changes as determined by ATR-FTIR and wettability nor protein content of the shells. Thus the reduction in mechanical properties is associated with breast implant failure but further research is required to elucidate the mechanisms.

The Effect of Eye Drop Excipients against Acanthamoeba Polyphaga by Alamar Blue TM Assay

The Effect of Eye Drop Excipients against Acanthamoeba Polyphaga by Alamar Blue TM Assay

Functionalization of silk fibroin through anionic fibroin derived polypeptides

Event Abstract Back to Event Functionalization of silk fibroin through anionic fibroin derived polypeptides Gabriele Griffanti1, 2, Mark James-Bhasin1, Ilaria Donelli3, Giuliano Freddi3 and Showan Nazhat1 1 McGill University, Department of Mining and Materials Engineering, Canada 2 Universita degli Studi di Milano, Dipartimento di Biotecnologie e Bioscienze, Italy 3 Stazione Sperimentale per la Seta, Italy Introduction: Silk fibroin (SF) is frequently considered as a template for mimicking biomineralization[1]. The α-chymotrypsin digestion of aqueous SF solutions generates anionic fibroin derived polypeptides (Cs)[2], which rapidly induce carbonated-hydroxyapatite (CHA) formation in dense collagen gels[3],[4]. Herein, it is hypothesized that the incorporation of Cs into SF would induce CHA deposition. The effect of Cs incorporation on the mineralization of electrospun (ES) SF scaffolds was assessed in simulated body fluid (SBF), and its potential role in mediating the osteoblastic differentiation of seeded mesenchymal stem cells (MSCs) was investigated. Materials and Methods: Cs [2] incorporated ES-SF scaffolds (at 1, 2, 5 and 25 wt.% Cs) were produced[5]. Zeta potential and contact angle measurements were used to investigate the effect of Cs on scaffold surface charge and hydrophilicity. Mineralization of the various scaffolds was assessed at days 1, 3 and 7 in 1.5 X Kokubo’s SBF[6]. Mineralization extent was assessed using ATR-FTIR, XRD and SEM. Passage 9 murine bone marrow derived MSCs were seeded at a density of 5x103 cells/cm2 on ES-SF scaffolds and cultured for 21 days in basal and osteogenic media. Alamar BlueTM assay was carried out to investigate seeded MSC proliferation at days 1, 7, 14 and 21 in culture. Quantitative real-time PCR was carried out at days 14 and 21 to assess MSC osteoblastic differentiation. Results: There was an increase in the negative charge properties of the ES-SF scaffolds with increasing Cs content (FIG 1, A). Immersion in SBF indicated CHA formation, increasing with Cs content (Fig. 1, B-C). Seeded MSCs attached on all scaffold types (Fig. 2) with differences in metabolic activities when cultured in osteogenic medium. MSCs on Cs incorporated ES-SF scaffolds demonstrated higher metabolic activities compared to those seeded on neat ES-SF (Fig. 3, A-B). Relative to basal medium, there was an up-regulation of alkaline phosphatase, Runx2 and Osteocalcin in osteogenic medium (at days 14 and 21). Surprisingly, under basal medium, MSCs seeded on Cs incorporated ES-SF scaffolds also expressed both Runx2 and Osteocalcin (FIG. 3, C-E). Discussion: In order for the onset of mineralization to occur, complexes such as –COOCa+ and (–COO)2Ca are required to form on the surface of SF. It is speculated that the carboxyl groups provided by the incorporation of negatively charged Cs polypeptides into SF served as nucleation sites for CHA deposition. Furthermore, Cs incorporation could be considered as a novel route to modulate MSC osteoblastic differentiation in the absence of osteogenic conditioning. Conclusions: The ability to incorporate Cs during SF fibre formation can induce CHA formation and control MSC osteoblastic differentiation, providing a basis for future optimization of ES-SF scaffolds for potential bone tissue engineering applications. Funding of CIHR, NSERC, CFI, Quebec MEIE, McGill University Faculty of Engineering Gerald Hatch Faculty Fellowship, MEDA and Università degli studi di Milano - Bicocca in collaboration with Assolombarda are gratefully acknowledged.

Assessment of Hygienic Effects of Laundering Using Spectrophotometry

Microbial cleanliness of textiles is an important issue in numerous industry sectors where microorganisms may contaminate manufactured goods and constitute a threat for consumers’ health. The most common method of removing contaminants like microbes is laundering. Changes in the manner of laundering, which have taken place in the recent years due to ecological reasons, lead to decreased hygienic effects. The aim of the study presented in this paper was to determine the viability of bacteria in textiles following laundering with the use of the spectrophotometric technique. The tests were conducted on cotton textiles contaminated with bacteria and laundered. The concentration of bacteria was determined by spectrophotometry with the use of AlamarBlue TM dye. The quantitative analyses confirmed the possibility of using spectrophotometric measurements in determining the levels of the tested bacterial species in the extracts from the laundered textiles. It was demonstrated that laundering textiles contaminated with bacteria result in a decrease in their count irrespective of the species.

One-pot synthesis of cinnamylideneacetophenones and their in vitro cytotoxicity in breast cancer cells

A series of cinnamylideneacetophenones were synthesized via a modified Claisen–Schmidt condensation reaction and evaluated for cytotoxicity against breast cancer cells using the Alamar Blue™ assay. Derivatives 17 and 18 bearing a 2-nitro group on the B ring, exhibited sub-micromolar cytotoxicity in MCF-7 cells (IC50=71 and 1.9nM), respectively. Derivative 17 also displayed sub-micromolar (IC50=780nM) cytotoxicity in MDA-MB-468 cells. Additionally, 17 and 18 displayed significantly less cytotoxicity than the chemotherapeutic doxorubicin in non-tumorigenic MCF-10A cells. This study provides evidence supporting the continued development of nitro-substituted cinnamylideneacetophenones as small molecules to treat breast cancer.

Fabrication and characterisation of PCL and PCL/PLA scaffolds for tissue engineering

Purpose – The main purpose of this research work is to study the effect of poly lactic acid (PLA) addition into poly (e-caprolactone) (PCL) matrices, as well the influence of the mixing process on the morphological, thermal, chemical, mechanical and biological performance of the 3D constructs produced with a novel biomanufacturing device (BioCell Printing). Design/methodology/approach – Two mixing processes are used to prepare PCL/PLA blends, namely melt blending and solvent casting. PCL and PCL/PLA scaffolds are produced via BioCell Printing using a 300-μm nozzle, 0/90° lay down pattern and 350-μm pore size. Several techniques such as scanning electron microscopy (SEM), simultaneous thermal analyzer (STA), nuclear magnetic resonance (NMR), static compression analysis and Alamar BlueTM are used to evaluate scaffold's morphological, thermal, chemical, mechanical and biological properties. Findings – Results show that the addition of PLA to PCL scaffolds strongly improves the biomechanical performance of the constructs. Additionally, polymer blends obtained by solvent casting present better mechanical and biological properties, compared to blends prepared by melt blending. Originality/value – This paper undertakes a detailed study on the effect of the mixing process on the biomechanical properties of PCL/PLA scaffolds. Results will enable to prepare customized PCL/PLA scaffolds for tissue engineering applications with improved biological and mechanical properties, compared to PCL scaffolds alone. Additionally, the accuracy and reproducibility of by the BioCell Printing enables to modulate the micro/macro architecture of the scaffolds enhancing tissue regeneration.

Nitric oxide-eluting nanocomposite for cardiovascular implants

Cardiovascular implants must resist thrombosis and intimal hyperplasia, but they are prone to such patency limiting conditions during graft implantation and prior to endothelialisation. Nitric oxide (NO) released from the endothelium has a complex protective role in the cardiovascular system, and this study has addressed: (1) in situ NO release profiles from S-nitrosothiols ((S-Nitroso-N-acetylpenicillamine (SNAP) and (S-Nitrosoglutathione (GSNO)) incorporated into polyhedral oligomeric silsesquioxanepoly(carbonate-urea)urethane (POSS-PCU) coronary artery bypass grafts (CABG) in a physiological pulsatile flow, and (2) the determination of their interaction with endothelial progenitor cells (EPCs), smooth muscle cells, platelets, whole blood kinetics. It was found that 1, 2, and 3wt% SNAP/GSNO incorporated into POSS-PCU-CABG successfully eluted NO, but optimal elution was evident with 2%-SNAP-POSS-PCU. NO release determined under static conditions using the Griess assay, and in situ measurements under pulsatile flow using amperometric probe was found to differ, thus confirming the significance of monitoring NO-elution under haemodynamic conditions.2%-SNAP-POSS-PCU demonstrated anti-thrombogenic kinetics through thromboelastography measurements, while metabolic activity using Alamar Blue™ assay and scanning electron microscopy demonstrated greater adhesion of EPCs and reduced adhesion of platelets.

English

Different fractions of&nbsp;Berberis glaucocarpa&nbsp;Stapf were tested for their antibacterial and anti-trypanosomal properties using the well established Alamar blueTM&nbsp;96 well microplate assays. Then eight known compounds (1 to 8), including five alkaloids (1 to 5), were isolated and characterized from this plant for the first time. All the compounds were tested for their antimicrobial and antitrypanosomal properties. Most of the pure compounds were also found to possess good antibacterial and anti-trypanosomal properties. Berberine (minimum inhibitory concentration (MIC) = 12.5&nbsp;&micro;g/ml), berberine chloroform (MIC = 25&nbsp;&micro;g/ml)&nbsp;and syringresinol (MIC = 12.5&nbsp;&micro;g/ml)&nbsp;were found to have anti-bacterial and anti-trypanosomal properties. The MIC values were determined for only active compounds. &nbsp; Key words:&nbsp;&nbsp;Berberis glaucocarpa,&nbsp;alkaloids, anti-trypanosomal, antibacterial.

Antiproliferative activity of Rhinella marina and Rhaebo guttatus venom extracts from Southern Amazon

The venom of amphibians is a fascinating source of active substances. In view of their medical importance and aiming to explore the amazing Brazilian biodiversity, we conducted bioprospecting of antiproliferative activity in extracts of Rhinella marina and Rhaebo guttatus toads occurring in the Southern Amazon of Mato Grosso, Brazil. LC–MS and HPLC analysis of the venom extracts of R. marina revealed four bufadienolides (telocinobufagin – 1, marinobufagin – 2, bufalin – 3 and resibufogenin – 4). R. guttatus venom extracts contained only marinobufagin (2). First, R. marina and R. guttatus venom extracts were evaluated for cytotoxicity against tumor cell lines by the MTT assay. All extracts revealed cytotoxicity, where R. marina extracts were comparable to doxorubicin (IC50 values ranging from 0.01 to 0.23 μg/mL). Only extracts of R. guttatus toad venom caused membrane disruption of human erythrocytes. The extracts were investigated for selective activity by determining their effect on stimulated human peripheral blood mononuclear cells (PBMC) with the Alamar Blue™ assay. The extracts were up to 80-fold more selective against leukemia cells when compared to dividing leukocytes. Aiming to confirm these antiproliferative effects, BrdU incorporation into DNA was measured in HL-60 treated cells with R. marina venom extracts. These extracts decreased BrdU incorporation at both concentrations tested. In summary, nine extracts of R. marina and R. guttatus venom showed pronounced lethal and discriminating effects on tumor lines, especially those from R. marina, highlighting toad parotoid gland secretions as a promising source for novel lead anticancer chemicals.

Silicon carbide quantum dots for bioimaging

Abstract

Proliferation and Osteoblastic Differentiation of hMSCs on Cellulose-Based Hydrogels

The aim of this project was to study the proliferation and differentiation of human Mesenchymal Stem Cells (hMSCs) onto a cellulose-based hydrogel for bone tissue engineering. Modified-cellulose hydrogel was prepared via double esterification crosslinking using citric acid. The response of human Mesenchymal Stem Cells (hMSCs) in terms of cell proliferation and differentiation into osteoblastic phenotype was evaluated by using Alamar blue assay and Alkaline phosphatase activity. The results showed that CMCNa and CMCNa_CA have no negative effect on hMSC, adhesion and proliferation. Moreover, the increase of the ALP expression for CMCNa_CA confirms the ability of the hydrogels to support the osteoblastic differentiation. The cellulose-based hydrogels have a potential application as filler in bone tissue regeneration.

Cytotoxic effects of 109 reference compounds on rat H4IIE and human HepG2 hepatocytes. III: Mechanistic assays on oxygen consumption with MitoXpress and NAD(P)H production with Alamar Blue™

In vitro toxicity screening can reduce the attrition rate of drug candidates in the pharmaceutical industry in the early development process. The focus in this study is to compare the sensitivity for cytotoxicity of a time-resolved fluoro metric oxygen probe with that of a fluoro metric Alamar Blue™ (AB) assay. Both assays measure mitochondrial activity by either oxygen consumption (LUX-A65N-1 (MitoXpress, Luxcel) probe) or NADH/FADH conversion (AB). Both assays were carried out with increasing concentrations of 109 reference compounds using rat H4IIE and human HepG2 hepatocytes at incubation periods of 24, 48 and 72h. Prior to this study, the influence on medium with either glucose or galactose was studied to analyze the rate of glycolysis and oxygen consumption, which latter process may be impaired in hepatoma cells. Inhibitors of oxygen consumption in combination with a glucose up-take inhibitor showed the largest consumption rate differences in the presence of 5mM of glucose.The choice for the 109 reference compounds was based on the so-called Multicentre Evaluation for In vitro Cytotoxicity (MEIC) and on diverse drug categories. For 59 toxic reference compounds, an evaluation for both assays was carried up to 10−3M. Toxicity was demonstrated with MitoXpress for 23 (39%) and 36 (61%) compounds in H4IIE and HepG2 cells, respectively, and with AB for 44 (75%) and 40 (68%) compounds. For 50 more pharmaceutical drugs more physiological concentrations were used up to 3.16×10−5M, and only 19 (38%) of these compounds appeared to be toxic in both assays.In conclusion, overall 63 (58%) and 60 (55%) compounds showed toxic effects with the MitoXpress and AB assays on rat H4IIE and human HepG2 hepatocytes, respectively. AB assays were more sensitive with respect to H4IIE cells and MitoXpress assays with respect to HepG2 cells. At all tested time intervals, MitoXpress showed its sensitivity, while AB is more sensitive at 48 and 72h. With AB more toxic compounds were identified, whereas MitoXpress was more sensitive for a few compounds. A species specific difference was clearly found with digoxin, a human specific potassium channel inhibitor. Thus both assays are valuable identifiers of early toxicity with discrimination in time, compounds and species.

Abstract A5: Cytotoxic Extracts of Ethnomedicinal Plants: Podophyllum peltatum (Mayapple) and Echinacea angustifolia

Abstract Phytochemical and other natural compounds are historically established as cures for many ailments in rural America and other developing countries. Our lab is testing plants that show potential for inhibiting the growth of cancer cells. The family Asteraceae and the family Berberidacae are very well known for their values as medicinal plants. More specifically, the genus species Echinacea angustifolia and genus species Podophyllum peltatum commonly known as the Mayapple, both are noted for their unique medicinal purposes. The crude methanol extracts of these plants were tested against human metastatic cell lines representing breast (MCF-7), (BT-549) and colon (SW620) tissues. Trypan blue exclusion is used to determine viable cell counts. Alamar BlueTM and florescent analysis were used to evaluate the cytoxicity of the extracts. Analysis of the crude extracts of Echinacea angustifolia has shown on MCF-7, BT-549, and SW620 that most of them exhibit anticancer activity against the cell lines at the maximum concentration of 200 μg/mL. In particular, less diluted concentrations have demonstrated exceptional inhibitory effects with IC50 values ranging between 0.10–15 μg/mL, respectively. On the other hand, analysis of the crude extracts of Podophyllum peltatum (Mayapple) has shown on MCF-7, BT-549 and SW620 some anticancer activity against cell lines at the maximum concentration of 200 μg/mL. More specifically, the more diluted concentrations have demonstrated exceptional growth inhibition with IC50 values ranging between 2–8 μg/mL Citation Format: Olena T. James, E. Lewis Myles. Cytotoxic Extracts of Ethnomedicinal Plants: Podophyllum peltatum (Mayapple) and Echinacea angustifolia [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer; 2012 Jan 8-11; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(2 Suppl):Abstract nr A5.

Antitrypanosomal Activity of Extract Fractions of Cetraria Islandica

The antitrypanosomal activity of various extract fractions of C. islandica (Iceland moss) was evaluated by using the well-established Alamar blue™ 96 well microplate assay. The antitrypanosomal activity significantly increased with increasing amounts of fractions (from 10-20 µg/ml) and MIC of 12.5µg/ml were observed for certain fractions. The results obtained in the present study indicate that C. islandica is a potential source of natural antitrypanosomal agents. © 2011 IGJPS. All rights reserved.