An in vitro test system for differentiation between antiproliferative and toxic effects in vascular smooth muscle cells.
We describe an in vitro test system looking for four endpoints in vascular smooth muscle cells (SMC): toxicity (cell number), DNA synthesis, reversibility of effects, and specificity of effects for SMC. SMC cultures at a low cell density, either actively proliferating or arrested by serum starvation and stimulated with 10% or 2% fetal calf serum (FCS) in parallel to treatment, are subject to a 52-h treatment phase with a test compound. Cultures were labeled with bromodeoxyuridine during the last 4 h of the treatment phase. Thereafter, the cultures are divided into two groups. In the first group, directly following treatment with a test compound, the cell number of the cultures is determined indirectly by using the vital dye neutral red. Subsequently, in the same cultures DNA synthesis is measured with an antibody directed to bromodeoxyuridine. The second group of cultures is reincubated with normal growth medium without test drug for a further 24 h. This recovery period is followed by determination of cell number and DNA synthesis as described above. This procedure allows determination of reversibility of effects observed directly after treatment giving important information for differentiation between toxic and antiproliferative mechanisms resulting in the reduction of DNA synthesis.
Herrmann
C
C
TA Cardiovascular Agents, Hoechst Marion Roussel, Frankfurt/Main, Germany. Claudia.Herrmann@hmrag.com
Cimiotti-Kolb
G
G
Lang
K
K
D000818:Animals; D001011:Aorta / Q000166:cytology; D002417:Cattle; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D004247:DNA / Q000096:biosynthesis / Q000187:drug effects; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects*; D020030:Nitric Oxide Donors / Q000494:pharmacology* / Q000633:toxicity*
D020030:Nitric Oxide Donors; D004247:DNA