Activation markers and cell proliferation as indicators of toxicity: A flow cytometric approach

Cell proliferation is an attractive endpoint inin vitro toxicity assays, since nearly any kind of damage in a cell may result in altered cell proliferation. In toxicological applications, liquid scintillation counting, measuring radioactivity from tritiated thymidine, has been the traditional way to estimate cell proliferation. An alternative approach is the measurement of BrdU incorporation by flow cytometry. Before the actual DNA synthesis starts, several proteins are expressed on the cell surface, as well as intracellularly. Among the markers on the cell surface CD69, CD25, and CD71 are sequentially expressed on human lymphocytes after a mitogenic stimulation. The aim of this study was to evaluate information obtained by analysis of expression of activation markers on cell surfaces in lymphocyte subsets and to compare it with data from cell proliferation studies performed by liquid scintillation counting and BrdU flow cytometry. The experiments were performed with phytohemagglutinin-stimulated human lymphocytes exposed to ochratoxin A and cyclosporin A. While ochratoxin A-treated cultures showed a steep inhibition with increasing concentration, the cyclosporin A treatment gave an inhibition curve with a less steep slope. Activation marker studies showed that the effect of treatment with both of the toxins was more pronounced on the late markers CD25 and CD71, while CD69 had the advantage that significant effects could be detected as early as 6 h after ochratoxin A treatment. Cyclosporin A treatment induced only minor alterations in CD69 expression. Certain differences in expression of activation markers between CD4⁺ and CD8⁺ subsets were found both in ochratoxin A- and cyclosporin A-treated cultures. A stimulating effect was found in cell cultures exposed to the lowest concentration of ochratoxin A on CD69 and CD25 expression. Signs of an increase in frequencies of proliferating cells measured with the BrdU flow cytometry method were also seen. This increase could not be detected with liquid scintillation counting. No other differences between the liquid scintillation counting and BrdU flow cytometry measurements of proliferation were obtained. We conclude that studies of activation marker expression by the flow cytometric approach used in this report are useful complements to traditional measurements of cell proliferation as they yield subsetspecific information about cellular processes which precede proliferation of lymphocytes.Abbreviations A pulse area - BrdU bromodeoxyuridine - CD cluster of differentiation - FBS fetal bovine serum - FITC fluorescein isothiocyanate - FL fluorescence - FSC forward light scatter - H pulse height - PBS phosphate-buffered saline - PI propidium iodide - R-PE R-phycoerythrin - RPMI Roswell Park Memorial Institute - SEM standard error of the mean - SSC orthogonal light scatter - W pulse width