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Vitro Screening (vitro + screening)
Selected AbstractsEstrogenic effect of leachates and soil extracts from lysimeters spiked with sewage sludge and reference endocrine disruptersENVIRONMENTAL TOXICOLOGY, Issue 2 2002Halim Dizer Abstract Several experiments were conducted to evaluate the behavior and performance of some potential endocrine disrupters (ECDs). Two in vitro screening assays, one based on MCF7-cell proliferation (E-screen test) and the other on estrogenic receptor activity [enzyme-linked receptor assay (ELRA)], were used for the tests, which were done in lysimeters 80 cm in diameter with depths of 30 cm (shallow) or 90 cm (deep). A sandy soil was used to fill in all lysimeters, which were spiked on the surface with either: (a) a sewage sludge (SS) at a dose equivalent to 20 tons ha,1; (b) a mixture of reference ECDs, comprising 17,- and 17,-estradiol (E2), nonylphenol, octylphenol, and bisphenol A at doses 100 times higher than the maximum concentrations respectively found in the applied SS; or (c) a mixture of ECDs and SS. After percolation of the lysimeters with rain and/or artificial water, five leachates were sampled from each lysimeter during a period of 210 days. Immediately after the lysimeter percolation experiments, four and six soil fractions were dissected from, respectively, the 30-cm and 90-cm lysimeters and extracted by water. Both the leachate and soil extract samples were analyzed for their estrogenicity using the assays indicated above. The E-screen assay was highly sensitive only for some leachate and extract samples but gave no response for most leachates and soil extracts. The results of the ELRA assay suggests a significantly higher estrogenicity of leachate samples from shallow lysimeters compared with that of leachates from deep lysimeters. In contrast, the estrogenic effect measured for soil extracts of shallow lysimeters was lower than that measured for soil extracts of deep lysimeters. The results of the E-screen assay suggests the occurrence of a fast mobilization of applied ECDs and a moderate retardation effect of native ECDs contained in applied SS in the sandy soil used in the lysimeters. In lysimeters spiked with a mixture of SS and ECDs, the washing-out effect of ECDs in the first leachate fraction decreased, but the distribution of ECDs in the lysimeters increased. The relatively high estrogenic impact measured for soil water extracts suggests that the ECDs were mostly associated with water-soluble fractions of organic matter and/or water-suspended fractions of the mineral soil matrix. The application of SS to agricultural and forest fields may determine the immobilization of ECDs in soil or their movement to surface and/or groundwater. Therefore, an endocrine risk of exposure exists for the water and soil organisms. © 2002 Wiley Periodicals, Inc. Environ Toxicol 17: 105,112, 2002; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/tox.10038 [source] Adhesion of perichondrial cells to a polylactic acid scaffoldJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 4 2003Alexander Giurea Abstract The number of chondrogenic cells available locally is an important factor in the repair process for cartilage defects. Previous studies demonstrated that the number of transplanted rabbit perichondrial cells (PC) remaining in a cartilage defect in vivo, after being carried into the site in a polylactic acid (PLA) scaffold, declined markedly within two days. This study examined the ability of in vitro culture of PC/PLA constructs to enhance subsequent biomechanical stability of the cells and the matrix content in an in vitro screening assay. PC/PLA constructs were analyzed after 1 h, 1 and 2 weeks of culture. The biomechanical adherence of PC to the PLA scaffold was tested by subjecting the PC/PLA constructs to a range of flow velocities (0.25,25 mm/s), spanning the range estimated to occur under conditions of construct insertion in vivo. The adhesion of PC to the PLA carrier was increased significantly by 1 and 2 weeks of incubation, with 25 mm/s flow causing a 57% detachment of cells after 1 h of seeding, but only 7% and 16% after 1 and 2 weeks of culture, respectively (p > 0.001). This adherence was associated with marked deposition of glycosaminoglycan and collagen. These findings suggest that pre-incubation of PC-laden PLA scaffolds markedly enhances the stability of the indwelling cells. © 2003 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved. [source] Extrapolating in vitro metabolic interactions to isolated perfused liver: Predictions of metabolic interactions between R -bufuralol, bunitrolol, and debrisoquineJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 10 2010Sami Haddad Abstract Drug,drug interactions (DDIs) are a great concern to the selection of new drug candidates. While in vitro screening assays for DDI are a routine procedure in preclinical research, their interpretation and relevance for the in vivo situation still represent a major challenge. The objective of the present study was to develop a novel mechanistic modeling approach to quantitatively predict DDI solely based upon in vitro data. The overall strategy consisted of developing a model of the liver with physiological details on three subcompartments: the sinusoidal space, the space of Disse, and the cellular matrix. The substrate and inhibitor concentrations available to the metabolizing enzyme were modeled with respect to time and were used to relate the in vitro inhibition constant (Ki) to the in vivo situation. The development of the liver model was supported by experimental studies in a stepwise fashion: (i) characterizing the interactions between the three selected drugs (R -bufuralol (BUF), bunitrolol (BUN), and debrisoquine (DBQ)) in microsomal incubations, (ii) modeling DDI based on binary mixtures model for all the possible pairs of interactions (BUF,BUN, BUF,DBQ, BUN,DBQ) describing a mutual competitive inhibition between the compounds, (iii) incorporating in the binary mixtures model the related constants determined in vitro for the inhibition, metabolism, transport, and partition coefficients of each compound, and (iv) validating the overall liver model for the prediction of the perfusate kinetics of each drug determined in isolated perfused rat liver (IPRL) for the single and paired compounds. Results from microsomal coincubations showed that competitive inhibition was the mechanism of interactions between all three compounds, as expected since those compounds are all substrates of rat CYP2D2. For each drug, the Ki values estimated were similar to their Km values for CYP2D2 indicative of a competition for the same substrate-binding site. Comparison of the performance between the novel liver physiologically based pharmacokinetic (PBPK) model and published empirical models in simulating the perfusate concentration,time profile was based on the area under the curve (AUC) and the shape of the curve of the perfusate time course. The present liver PBPK model was able to quantitatively predict the metabolic interactions determined during the perfusions of mixtures of BUF,DBQ and BUN,DBQ. However, a lower degree of accuracy was obtained for the mixtures of BUF,BUN, potentially due to some interindividual variability in the relative proportion of CYP2D1 and CYP2D2 isoenzymes, both involved in BUF metabolism. Overall, in this metabolic interaction prediction exercise, the PBPK model clearly showed to be the best predictor of perfusate kinetics compared to more empirical models. The present study demonstrated the potential of the mechanistic liver model to enable predictions of metabolic DDI under in vivo condition solely from in vitro information. © 2010 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:4406,4426, 2010 [source] Evaluation of human nasal RPMI 2650 cells grown at an air,liquid interface as a model for nasal drug transport studiesJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 3 2008Shuhua Bai Abstract This study tests the hypothesis that human nasal RPMI 2650 cells grown at an air,liquid interface is a feasible model for drug transport studies via the nasal route. RPMI 2650 cells were cultured in Eagle's minimal essential medium (MEM) at both air,liquid and liquid,liquid interfaces. For each culture regimen, monolayer integrity was tested by measuring the transepithelial resistance (TEER) as well as the transport of paracellular and transcellular markers across the monolayer. The expression of tight junction proteins,differentiation markers,in cells of the different monolayers was studied by western blot analysis and confocal microscopy. The highest TEER values (192,±,3 ,,·,cm2) were observed for RPMI 2650 cells seeded onto collagen-coated permeable polytetrafluoroethylene inserts and grown at an air,liquid interface for 10 days; a seeding density of 4,×,105/cm2 generated and maintained a cell monolayer with suitable barrier properties at days 9,12. Microscopic examination showed that RPMI 2650 cells grown on filter inserts formed a fully confluent monolayer. The apparent permeability coefficients of the paracellular marker, [14C] mannitol, and the transcellular marker, [3H] propranolol, were 5.07,±,0.01,×,10,6 cm/s and 16.1,±,0.1,×,10,6 cm/s, respectively. Western blot analysis indicated the presence of four tight junction proteins: ZO-1, occludin, claudin-1 and E-cadherin; and the quantities of ZO-1, occludin, and E-cadherin were significantly higher in cells grown at an air,liquid interface than in cells grown at a liquid,liquid interface. Confocal microscopic studies showed ZO-1, F-actin, occludin and claudin-1 proteins at cell-cell contacts and revealed significant differences in the distributions and densities of ZO-1 protein in cells grown at the two types of interface. The data indicate that RPMI 2650 cells grown at an air,liquid interface form polarized monolayers with the cells interconnected by tight junction proteins. This human nasal cell line model could provide a useful tool for in vitro screening of nasal drug candidates. © 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 97:1165,1178, 2008 [source] Rapid Screening Method of Cassava Cultivars for Resistance to Colletotrichum gloeosporioides f.sp. manihotisJOURNAL OF PHYTOPATHOLOGY, Issue 1 2002C. N. FOKUNANG An in vitro method for assessing cassava anthracnose disease (CAD) resistance was developed as a preliminary screen to a CAD-resistant breeding programme. Potato dextrose agar (PDA) media was amended by extracts from the stem cortex of 10 cassava cultivars (30001; 30572, 30211, 88/02549, 88/00695, 88/01336, 91/00344, 91/00313, 91/00684 and 91/00475), and assayed for efficacy of inhibition of the growth of Colletotrichum gloeosporioides f. sp. manihotis isolates (05FCN, 10FCN, 12FCN, and 18FCN). Morphological and physiological data indicated that there was a significant difference (P , 0.05), in mycelial growth, spore germination and sporulation among the four isolates on PDA amended with cassava stem extracts. Extracts from cassava cultivars 30211, 91/00684 and 91/00313 showed higher inhibition of germ tube development, mycelial growth and sporulation of the fungal isolates, whereas cultivars 88/02549 and 88/01336 showed the least inhibition. The 10 cultivars were further tested in both greenhouse and field conditions, under disease pressure for two planting seasons, to corroborate resistance to the fungus as observed in vitro. Greenhouse and field trials with the 10 cassava cultivars showed a significant difference (P , 0.05) in CAD resistance. Cultivars 88/02549 and 88/01336 were highly CAD-susceptible, as shown in the in vitro assays and confirmed in the greenhouse and field tests. The other eight cultivars were either resistant (30211, 91/00684), or moderately resistant (30572, 88/00695, 91/00475, 91/00344, 30001 and 91/00313) to CAD. The study shows that an in vitro screening assay of cassava for resistance to CAD could serve as a convenient preliminary screening technique to discriminate CAD-resistant from CAD-susceptible cassava cultivars. The in vitro screening method considerably reduces time and labour in comparison with the current screening techniques of cassava, which involve field planting, inoculation and evaluation. [source] | ||||||||||