A two-parameter flow cytometry protocol for the detection and characterization of the clastogenic, cytostatic and cytotoxic activities of chemicals.
Cultured, freshly-isolated rat fibroblasts were exposed in vitro to vincristine sulphate (VC), amethopterin (AM), bleomycin (BL), benomyl (BE) and practolol (PR). Cells treated for 5 h were subjected 24 h later to a two-parameter (DNA/protein) flow cytometry analysis. The fluorochromes used were sulphorhodamin 101 and DAPI. From DNA and protein histograms, alterations in cell-cycle kinetics, variations in the amount of DNA in individual G1-phase cells and the enhancement of or increased variation in the protein content of the exposed cells were determined. Each of the 5 chemicals induced a specific dose-dependent pattern of changes in the DNA and protein histograms. DNA dispersion was enhanced with VC, AM, BL and BE but not with PR. The cell cycle was blocked in the G2 phase with VC, at early S phase with amethopterin and, depending on the dose, at the G1 or G2 phase with bleomycin or at the S phase or G2 phase with benomyl. Practolol inhibited cells slightly in the S phase at the highest exposure level. Protein analysis allows cytotoxic activity (loss of proteins) or induced unbalanced growth (protein accumulation) of test compounds to be recognized. The results obtained imply that the proposed two-parameter DNA/protein analysis by flow cytometry is a suitable method for prospective testing of chemicals for their induction of structural or numerical chromosome aberrations. Simultaneously, a broad range of cytotoxic, cytostatic and cell-cycle perturbing activities of the test agents can be recognized.
D000818:Animals; D001542:Benomyl / Q000633:toxicity; D001761:Bleomycin / Q000633:toxicity; D002455:Cell Division / Q000187:drug effects; D004247:DNA / Q000032:analysis; D005434:Flow Cytometry / Q000379:methods; D006099:Granuloma / Q000473:pathology; D008297:Male; D008727:Methotrexate / Q000633:toxicity; D009152:Mutagenicity Tests / Q000379:methods; D009153:Mutagens / Q000633:toxicity*; D009154:Mutation; D011217:Practolol / Q000633:toxicity; D011506:Proteins / Q000032:analysis; D051381:Rats; D011919:Rats, Inbred Strains; D014750:Vincristine / Q000633:toxicity
D009153:Mutagens; D011506:Proteins; D001761:Bleomycin; D014750:Vincristine; D004247:DNA; D011217:Practolol; D001542:Benomyl; D008727:Methotrexate