SIMULTANEOUS EVALUATION OF DEXAMETHASONE-INDUCED APOPTOSIS AND MICRONUCLEI IN RAT PRIMARY SPLEEN-CELL CULTURES

Apoptosis or programmed cell death is a biological event that is bioch emically and morphologically distinct from cellular necrosis. Nonethel ess, its relationship has not been studied in terms of a cytogenetic e ndpoint such as micronucleus formation. In the present study, based on cytological observations, the incidence of dexamethasone-induced apop totic cells was related to the frequency of micronucleated cells in vi tro. Rat primary spleen cells were grown in 6-well plates with RPMI 16 40 media using concanavalin A and lipopolysaccharide as mitogens. At c ulture initiation, the test agent dexamethasone (10, 20 or 40 mu M) an d a cytokinesis inhibitor cytochalasin B (3 mu g/ml) were added. Cultu res were harvested 18 h and 40 h later. Slides were prepared and stain ed with Diff-Quik stain. Frequencies of apoptotic cells and micronucle ated binucleate cells were enumerated cytologically based on 500 cells per treatment from the same slides. The results showed a dose-depende nt increase in the number of apoptotic cells in rat spleen cultures tr eated with dexamethasone. At 18 h, the percentages of apoptotic cells were 0.8, 1.6, 3.4 and 4.4 with 0, 10, 20 and 40 mu M dexamethasone, r espectively. The corresponding percentages of apoptotic cells at 40 h were: 2.8, 2.6, 5.6 and 10.4. However, at the same concentrations of d examethasone, the micronucleus frequency in binucleate cells remained relatively unchanged. The phenomenon of apoptosis induced by dexametha sone was confirmed biochemically based on a characteristic DNA 'ladder ' pattern by gel electrophoresis. These data suggest that dexamethason e at the concentrations which induced apoptosis did not produce cytoge netic damage. Also, these findings indicate that micronucleus formatio n and nuclear changes leading to apoptosis are separate events and the se endpoints may not be closely correlated for dexamethasone.