Alterations in the cellular DNA and protein content determined by flow cytometry as indicators for chemically induced structural and numerical chromosome aberrations

Abstract

Cellular DNA and protein content were determined simultaneously in freshly isolated fibroblast-like rat cells by flow cytometry. After exposure to doxorubicin, nitrofurantoin, propranolol and practolol at a low, tissue like oxygen concentration (5% O 2 ), drug-induced alterations in cell cycle kinetics, in the distribution of DNA and in the protein content of G,-phase cells (nucleus/cytoplasm ratio) were analysed. Optimal exposure time (5 or 24 h) and recovery interval (24 or 48 h) were determined. Variation in the exposure time and recovery period can affect cell cycle kinetics both qualitatively and quantitatively, whereas the distribution of DNA and protein content are affected quantitatively only. A 24-h exposure combined with a 24-h recovery period proved to be the most efficient approach. Each of the tested chemicals induced a specific, dose-dependent pattern of altered cellular DNA and protein content. Comparison with results obtained in other genotoxicity tests, and with data reported earlier, showed that this two-parameter protocol can be used to recognize and to characterize chemicals as clastogens, or as compounds with a combined cytostatic/clastogenic activity, or as spindle-poison-like compounds.

Effects of sodium butyrate on DNA content, glutathione S-transferase activities, cell morphology and growth characteristics of rat liver nonparenchymal epithelial cells in vitro

“…In that model the peak of chromosome distribution was widened, showing maxima of nuclei with 54, 61 and 66 chromosomes. Cells which had been exposed to butyrate for 3 days were analysed by two-parameter flow cytometry (28). Using this technique, similar effects were observed as in the long-term treatment model (data not shown).…”

Cellular heterogeneity of DNA/total‐protein content in human lung tumors, as determined by flow cytometry

“…We used the SR 101 as protein staining, as it had provided reasonably good results for others (Gohde and Dittrich, 1970;Stohr et al, 1978;Maier and Schawalder, 1988). However, some problems arise in using such a method.…”

Single cell analysis in toxicity testing: the mitogenic activity of thioacetamide in cultured rat hepatocytes analyzed by DNA/protein flow cytometry

“…Within 4-24 h after fixation, cells or nuclei were stained in a freshly mixed staining solution of 0.5 ml SR 101 (Sulforhodamine 101) stock solution (30 mg SRI01/100 ml TRIS buffer: for SR and GG455 filter for DAPI signals. Calibration and the measurement of cells from each dish (10-20 x 103 analyzable cells) were carried out as described elsewhere (Maier and Schawalder 1988). Data acquisition occurred in list mode and histograms obtained from gated areas were analyzed with the software ACAS (O. Ahrens, Bargteheide, BRD, 1990) on an IBM compatible computer (Olivetti M380XP1).…”

“…1) the number of cells in each degree of ploidy (2C, 4C, 8C including 6C aggregates) and in the S-phase (Table 2) was determined and combined with the analysis of DNA signal dispersions (coefficient of variations not shown) of cells and nuclei using the ACAS II software (Ahrens, Bargteheide, BRD); 2) a correction for aggregated cells and nuclei (see Table 2 and Appendix I) was made; 3) the contribution of binuclear cells was calculated from data obtained by nuclei analysis and with formulas described in Appendix II (Tables 3 and 4); 4) from protein histograms (Fig. 1) of gated TCs, the cellular protein content (Cp values: Maier and Schawalder 1988) including the dispersion of the protein signals (not shown) was determined (Tables 3 and 4).…”