Fachbereich Veterinärmedizin



    Neutral Rot-Aufnahmetest Modifikation, Weiterentwicklung und Standardisierung zur Bestimmung dreier Endpunkte (Zelladhäsion, -proliferation und –vitalität) in einem gemeinsamen Testansatz (2001)

    Sillinger, Astrid Marie-Luise
    Berlin, 2001 — XI, 185 Seiten
    Institut für Tierschutz und Tierverhalten

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    Gebäude 21, 1. OG
    14163 Berlin
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    email: tierschutz@vetmed.fu-berlin.de

    Abstract / Zusammenfassung

    In the first part of this investigation the attempt was made to modify a known cellular system for toxicity testing (Neutral Red Assay) by determining three endpoints instead of one singular endpoint (general cytotoxicity). These endpoints had to include the processes of cell adhesion, cell proliferation and cell vitality. On the basis of the cellular system for toxicity testing, as described by Borenfreund and Puemer (1985), we tried to vary individual steps in the proceedings of the method (number of cells at the beginning of the assay, duration of substance exposure, suspension scheme, duration of the culture period) and y doing so to specify the obtained results with regard to individual cell processes and finally to standardize the extended assay. The following conditions allowed the extension of the method by the mentioned endpoints:Number of cells at the beginning of the assay 5.000 cells per 0.2 nilDuration of substance exposure 6 hFumigation scheme (endpoint adhesion) 10 Vol% 02, 5 Vol% C02, 85 vol% N2Suspension culture (endpoint adhesion) 4 h in a "roller device" integrated in an incubatorDuration of culture period 6 h endpoint cell adhesion 33 h endpoint cell death 48 h endpoint cell prolif erationIn the second part of this investigation the developed modification was screened for its practicability and efficiency by employing model substances. We investigated the effects of cytochalasin B, colchicine and dimethylsulfoxide (DMSO) on cell adhesion, cell proliferation and cell vitality. In this modified system, cytochalasin B induced an inhibition of cell adhesion at 1 yg/ml which increased with increasing concentrations. Colchicine led to an inhibition of proliferation processes in the MDCK cell culture. This effect also first occurred after 1 yg colchicine / mI and increased with increasing concentrations. Treatment of the MDCK cells with the solvent DMSO led to general cell death within the culture at all concentrations tested. This effect was also more expressed with increasing concentrations. The application of model substances proved the efficiency as well as practicability of the system.The necessity of frequent morphological examinations and the lack of more detailed information on the modes of action of the individual test substances require, depending on the specific issue, more extensive or parallel additional test methods.In the third part, the effects of the test substances N-dimethylformamide (DMF) and the two metabolites S-(N-methylcarbamoyl)glutathione (SMG) and S-(N-methylcarbamoylcysteine (SMC) were assessed. The effects of these three substances had been tested in an other toxicity test system (lesions of mouse limb buds induced in an organ culture system). Comparison of the obtained results showed that DMF did not have any effects on cell adhesion and cell proliferation at any concentration applied, neither did it lead to cell death. In organ culture, it did not exhibit any effects either. Limb bud development was not affected at any concentration.SMC had a cytotoxic effect from 10 yg/ml on, resulting in cell death. With increasing concentrations a higher percentage of the cell population was affected. In organ culture, this substance led to a disturbance in limb bud development at doses from 100 yg/ml on. Increasing concentrations caused a higher degree of lesions. This substance exhibited the highest toxic potential in organ as well as in cell culture.SMG showed a medium toxic potential in the two culture systems. From 100 yg SMG / mI in medium onwards the Neutral Red Assay revealed a concentration-dependent cytotoxic effect that clearly increased with increasing concentrations. This result was in agreement with results obtained in organ culture where first lesions were observed in the form of reduced limb bud growth at a concentration of 100 yg SMG / m]. These lesions were most pronounced after 300 yg SMG / mI.The fourth part of this investigation includes the preparation of MDCK cells for flow cytometry. Using monoclonal antibodies (mAb), the following adhesion molecules could be demonstrated on the surface of untreated MDCK cells: antibodies against the B, -chain (CD29) and against the laminin receptor (a6ß1, CD49f) yielded the most intensive staining. The staining intensity was slightly weaker with the mAb against the fibronectin receptor (a4ß1), the hyaluronic acid receptor (CD44), and the ELAM receptor (CD15). There were some uncertainties with regard o the demonstration of the a2-, asand the ß3-chains. The use of mAb against these surface structures showed stronger as well as weaker staining and the reproducibility of the results was poor.Despite cell treatment with cytochalasin B, flow cytometric determinations proved the presence of the above-mentioned receptors. However, the expression of the a6-chain was reduced with increasing concentrations. This indicated a diminution of Epitopendichte. A relationship probably exists between the reduced adhesion capacity of the cells in the Neutral Red Assay (after cell treatment with cytochalasin B) and the reduced density of the receptors belonging to the adhesion molecules.