ATLA 26.3, May 1998

//ATLA 26.3, May 1998

Transgenic Animals: More than “Commercial Opportunities”

Michael Balls

It is very hard these days to escape from being all too regularly confronted with worrying prospects and problems and real or potential ethical indecencies resulting from “progress” in human and animal developmental biology and genetics. The production and use of transgenic animals has reversed
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2017-01-09T06:26:42+00:00 Tags: |

Reducing the Use of Laboratory Animals in Biomedical Research: Problems and Possible Solutions

Michael F.W. Festing, Vera Baumans, Robert D. Combes, Marlies Halder, Coenraad F.M. Hendriksen, Bryan R. Howard, David P. Lovell, Graham J. Moore, Philip Overend and Marie S. Wilson

This is the report of the twenty-ninth of a series of workshops organised by the European Centre for the Validation of Alternative Methods (ECVAM). ECVAM’s main goal, as defined in 1993 by its Scientific Advisory Committee, is to promote the scientific and regulatory acceptance of alternative methods which are of importance to the biosciences and which reduce, refine or replace the use of laboratory animals. One of the first priorities set by ECVAM was the implementation of procedures which would enable it to become well-informed about the state-of-the-art of non-animal test development and validation, and the potential for the possible incorporation of alternative tests into regulatory procedures.
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2017-01-09T06:26:42+00:00 Tags: , , |

A Comparison of the Acute and Chronic Effects of Antidepressants in Cultured C6 and 1321N1 Cells

N. Debbie Slamon and Vic W. Pentreath

The cytotoxicities of the antidepressants amitriptyline, imipramine (both tricyclic), fluoxetine (a selective serotonin re-uptake inhibitor) and tranylcypromine (a monoamine oxidase inhibitor) were compared in vitro in rat (C6) glioma and human (1321N1) astrocytoma cell lines. Differences in toxicity were determined after acute (24-hour) and chronic (7-day) administration and assessed by using the neutral red uptake (NRU) assay, the MTT assay, increased expression of glial fibrillary acidic protein (GFAp), and reactive morphology criteria. The relative toxicities (EC50 [concentration causing an effect in 50% of cells] value range) were fluoxetine > amitriptyline > imipramine > tranylcypromine for all the tests employed, in both cell lines and at both exposure times. There was a high and significant positive correlation between the different cell types, at both exposure times, with both the NRU and MTT assays. Increases in MTT reduction, NRU, and GFAp expression associated with cell activation were noted in C6 cells after exposure for 24 hours, but decreased after exposure for 7 days. For 1321N1 cells, increases in NRU were only observed after exposure for 24 hours. Reactive-type changes in morphology were seen after exposure to all the antidepressants, in both the C6 and 1321N1 cell lines. The data show that low concentrations of antidepressants induce metabolic changes in the astrocyte cell lines, with some significant differences in the patterns of toxicity of the tested substances.
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Direct Determination of Glutathione S-transferase and Glucose-6-phosphate Dehydrogenase Activities in Cells Cultured in Microtitre Plates as Biomarkers for Oxidative Stress

Concepción García-Alfonso, Guillermo Repetto, Pilar Sanz, Manuel Repetto and Juan López-Barea

The enzymes glutathione S-transferase (GST) and glucose-6-phosphate dehydrogenase (G-6PDH) are implicated in the defence against oxidative stress. GST is mainly involved in the conjugation of electrophilic compounds with glutathione (GSH), although some of its isoenzymes display peroxidase activity. G-6PDH and glutathione reductase regenerate NADPH and GSH, respectively, to restore the reduced intracellular redox status following oxidative stress. Enzymatic assays for GST and G-6PDH were adapted and optimised to permit the direct in vitro determination of the effects of toxicants which induce oxidative stress in cells on microtitre plates, thereby avoiding the need to prepare cell-free extracts. To optimise the conditions of the enzymatic assays, GST activity was measured at substrate concentrations of 1–3mM GSH and 1–3mM 1-chloro-2,4-dinitrobenzene, while G-6PDH activity was measured at 7.5–37.5mM glucose-6-phosphate and 55–275mM NADP. Both enzymatic activities were directly proportional to cell number up to a density of 1 × 105 cells/well. The effects on GST and G-6PDH activities of three toxicants which induce oxidative stress — paraquat, iron (II) chloride and iron (III) chloride — were compared in cultured Vero cells to validate the new assays. Specific GST activity increased to 145% and 171% compared to the controls in cells treated with 5mM paraquat and 5mM iron (II) chloride, respectively, but was inhibited after exposure to 25mM iron (III) chloride. Specific G-6PDH activity increased to 136% compared to the control after exposure to 5mM paraquat, but was inhibited in cells exposed to 5mM iron (II) chloride and 25mM iron (III) chloride.
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Toxicity and Cell Density Monitoring in Monolayer and Three-dimensional Cultures with the XTT Assay

Xavier Ponsoda, Maria José Gómez-Lechón and José V. Castell

The application of viability criteria (MTT and XTT tests) to monolayer cultures and immobilised cells in three-dimensional systems was investigated in order to assess cell viability and cell proliferation. The suitability and accuracy of these tests were compared with the conventional criteria (cellular protein and DNA content) used in monolayer cultures for the same purpose. The colorimetric assay based on the metabolic reduction of the tetrazolium salt XTT to a water-soluble formazan proved to be very useful, rapid and sensitive. This automated spectrophotometric enzymatic method, due to its lack of toxicity, also permits repeated nondestructive assays on a single cellular culture for the long-term monitoring of cytotoxicity, cell survival and cell proliferation, and can be performed in 96-well plates with minimal handling. This method could offer a solution for cellular density evaluation in complex cell cultures that do not permit visual examination; it is also the best choice for protein-based, three-dimensional systems such as collagen gels.
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Magnetic Resonance Imaging of the Common Marmoset Head

Vee-Meng Lee, Newman G. Burdett, T. Adrian Carpenter, Nicholas J. Herrod, Michael F. James and Laurance D. Hall

This study evaluated the changes in the intrinsic magnetic resonance (MR) relaxation parameter values (T1, T2, proton density, magnetisation transfer and apparent diffusion coefficient) of the marmoset head, imaged before and after death. Knowing the absolute values of the MR parameters makes it possible to choose an imaging protocol for optimal structural
differentiation. The changes between the ante-mortem and post-mortem MR parameters provide an insight into the changing biophysical microenvironment of the post-mortem brain, and allow some of the changes that occur in pathological conditions to be predicted. Diffusionweighted MR imaging (MRI) was used to map quantitative apparent diffusion coefficient values, and to investigate diffusional anisotropy along the fibre tracts in pre-mortem and post-mortem brain tissue. A three-dimensional data set of the entire marmoset brain demonstrates the ability of three-dimensional MRI to differentiate internal brain structures. MRI is a non-invasive technique which, in principle, permits the same animal to be re-imaged serially and has the potential to probe in vivo brain structural and biophysical changes over an extended period of time. Serial imaging, where each animal acts as its own control, reduces the number of animals required to detect a significant change by minimising the effects of inter-subject variance. MRI therefore provides important scientific and ethical benefits.
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