Publikationsdatenbank

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

Art

Hochschulschrift

Autor

Sillinger, Astrid Marie-Luise

Quelle

Berlin, 2001 — 185 Seiten

Kontakt

Institut für Pharmakologie und Toxikologie

Koserstr. 20
14195 Berlin
+49 30 838 53221
pharmakologie@vetmed.fu-berlin.de

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.