Centromere analysis of micronuclei induced by 2‐aminoanthraquinone in cultured mouse splenocytes using both a gamma‐satellite DNA probe and anti‐kinetochore antibody

Abstract

We have tested 2-aminoanthraquinone (2-AAQ) as a potential aneugen in a cytokinesis-blocked mouse splenocyte micronucleus (MN) assay. Binucleated cells (BNC) were evaluated for MN, and the MN were further probed with two indicators of centromere presence: an anti-kinetochore autoantibody and a DNA probe for the mouse gamma-satellite locus. A dose-dependent increase in the frequency of BNC with MN was observed. At the highest 2-AAQ concentration (10 micrograms/ml), the frequency of BNC containing MN was increased greater than 10-fold over background. Both centromere-positive and centromere-negative MN were significantly increased. At least 62% of MN at all 2-AAQ doses were positive for the gamma-satellite DNA probe, while 30-53% were labeled with the antikinetochore antibody. In contrast with the 2-AAQ results, after treatment with the aneugen demecolcine (positive control), greater than 80% of MN labelled positive with both probes. This discordance in the results with the 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 proteins, as a clastogen with a preferential cleavage site at or near the gamma-satellite locus, or both. Our results also suggest that the use of either of these probes individually may not be an adequate measure of centromere presence. Nevertheless, positive results for both markers provides strong evidence that 2-AAQ is aneugenic.