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 — 185 Seiten
    Institut für Pharmakologie und Toxikologie

    Koserstr. 20
    14195 Berlin
    +49 30 838 53221

    Abstract / Zusammenfassung

    In the first part of this investigation the attempt was made to modify a known c
    ellular system for toxicity testing (Neutral Red Assay) by determining three end
    points instead of one singular endpoint (general cytotoxicity). These endpoints
    had to include the processes of cell adhesion, cell proliferation and cell vital
    ity. On the basis of the cellular system for toxicity testing, as described by B
    orenfreund and Puemer (1985), we tried to vary individual steps in the proceedin
    gs of the method (number of cells at the beginning of the assay, duration of sub
    stance exposure, suspension scheme, duration of the culture period) and by 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 begin
    ning of the assay 5.000 cells per 0.2 nilDuration of substance exposure 6 hFumig
    ation scheme (endpoint adhesion) 10 Vol% 02, 5 Vol% C02, 85 vol% N2Suspension cu
    lture (endpoint adhesion) 4 h in a "roller device" integrated in an incubatorDur
    ation of culture periodi6 h endpoint cell adhesiont33 h endpoint cell deatho48 h
    endpoint cell prolifxerationIn the second part of this investigation the develo
    ped 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 als
    o first occurred after 1 yg colchicine / mI and increased with increasing concen
    trations. 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 p
    roved the efficiency as well as practicability of the system.The necessity of fr
    equent morphological examinations and the lack of more detailed information on t
    he modes of action of the individual test substances require, depending on the s
    pecific issue, more extensive or parallel additional test methods.In the third p
    art, the effects of the test substances N-dimethylformamide (DMF) and the two me
    tabolites S-(N-methylcarbamoyl)glutathione (SMG) and S-(N-methylcarbamoylcystein
    e (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 cu
    lture system). Comparison of the obtained results showed that DMF did not have a
    ny 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 eff
    ects 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 co
    ncentrations a higher percentage of the cell population was affected. In organ c
    ulture, this substance led to a disturbance in limb bud development at doses fro
    m 100 yg/ml on. Increasing concentrations caused a higher degree of lesions. Thi
    s substance exhibited the highest toxic potential in organ as well as in cell cu
    lture.SMG showed a medium toxic potential in the two culture systems. From 100 y
    g SMG / mI in medium onwards the Neutral Red Assay revealed a concentration-depe
    ndent cytotoxic effect that clearly increased with increasing concentrations. Th
    is result was in agreement with results obtained in organ culture where first le
    sions 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 f
    ourth 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 mA
    b against the fibronectin receptor (a4ß1), the hyaluronic acid receptor (CD44),
    and the ELAM receptor (CD15). There were some uncertainties with regard to the d
    emonstration of the a2-, asand the ß3-chains. The use of mAb against these surfa
    ce structures showed stronger as well as weaker staining and the reproducibility
    of the results was poor.Despite cell treatment with cytochalasin B, flow cytome
    tric determinations proved the presence of the above-mentioned receptors. Howeve
    r, the expression of the a6-chain was reduced with increasing concentrations. Th
    is indicated a diminution of Epitopendichte. A relationship probably exists betw
    een the reduced adhesion capacity of the cells in the Neutral Red Assay (after c
    ell treatment with cytochalasin B) and the reduced density of the receptors belo
    nging to the adhesion molecules.