CENTROMERE ANALYSIS OF MICRONUCLEI INDUCED BY 2-AMINOANTHRAQUINONE INCULTURED MOUSE SPLENOCYTES USING BOTH A GAMMA-SATELLITE DNA-PROBE ANDANTI-KINETOCHORE ANTIBODY

We have tested 2-aminoanthraquinone (2-AAQ) as a potential aneugen in a cytokinesis-blocked mouse splenocyte micronucleus (MN) assay. Binucl eated cells (BNC) were evaluated for MN, and the MN were further probe d with two indicators of centromere presence: an anti-kinetochore auto antibody and a DNA probe for the mouse gamma-satellite locus. A dose-d ependent increase in the frequency of BNC with MN was observed. At the highest 2-AAQ concentration (10 mu g/ml), the frequency of BNC contai ning MN was increased greater than 10-fold over background. Both centr omere-positive and centromere-negative MN were significantly increased . At least 62% of MN at all 2-AAQ doses were positive for the gamma-sa tellite DNA probe, while 30-53% were labeled with the anti-kinetochore antibody. In contrast with the 2-AAQ results, after treatment with th e aneugen demecolcine (positive control), greater then 80% of MN label led positive with both probes. This discordance in the results with th e two probes after 2-AAQ exposure suggests that the mode of action of this chemical may be as an aneugen by disruption of the kinetochore pr oteins, as a clastogen with a preferential cleavage site at or near th e gamma-satellite locus, or both. Our results also suggest that the us e of either of these probes individually may not be an adequate measur e of centromere presence. Nevertheless, positive results for both mark ers provides strong evidence that 2-AAQ is aneugenic. (C) 1994 Wiley-L iss, Inc.