Etoposide and merbarone are clastogenic and aneugenic in the mouse bone marrow micronucleus test complemented by fluorescence in situ hybridization with the mouse minor satellite DNA probe

Abstract

The topoisomerase II (topo II) inhibitors etoposide (VP-16) and merbarone (MER) were investigated with the in vivo micronucleus test (MN test) combined with fluorescence in situ hybridization (FISH) using the mouse minor satellite DNA probe to discriminate MN of clastogenic and aneugenic origin. All experiments were performed with male (102/ElxC3H/El) F1 mice bred in the mouse colony of the GSF Research Center. The sample size per experimental group was five animals and 2,000 polychromatic erythrocytes (PCE) were scored per animal from coded slides in the conventional MN test. A separate set of coded slides was used for the FISH analysis. All treatments consisted of single intraperitoneal injections. Colchicine (COL, 3 mg/kg) and mitomycin (MMC, 1 mg/kg) were used as a positive control aneugen and clastogen, respectively, and these compounds produced the expected responses. A dose of 1 mg/kg VP-16 induced 3.44% MNPCE (compared to the concurrent solvent control of 0.37%, P < 0.001) and of these 39.9% (1.4% MNPCE) showed one or more fluorescent signals. MER (7.5-60 mg/kg) increased the MNPCE frequencies in a dose-dependent manner, with 15 mg/kg being the lowest positive dose. At the highest dose of 60 mg/kg of MER, a total of 4.26% MNPCE were found (compared to 0.31% in the concurrent solvent control, P < 0.001) and of these 46.2% (2.0% MNPCE) contained one or more fluorescent signals. The data demonstrate that VP-16 and MER induced both clastogenic and aneugenic events despite their different modes of topo II inhibition.

The DNA topoisomerase II catalytic inhibitor merbarone is genotoxic and induces endoreduplication

“…It has been reported that merbarone is aneugenic as well as clastogenic [33]. In good agreement with our earlier reports on the effectiveness of different topo II inhibitors to induce endoreduplication [16,40], in the present investigation we have observed that merbarone also inhibits topo II catalytic activity in cultured Chinese hamster ovary AA8 cell line and induces endoreduplication under conditions where also DNA damage is induced.…”

“…Nevertheless, more recent investigations have shown that merbarone and the structurally related bisdioxopiperazines (ICRF-193, and ICRF-187) induced strong dose-dependent genotoxic effects in mammalian cells similar to those seen with the topo II poison etoposide. In addition, clastogenic effects could be seen in vivo in bone marrow erythrocytes from merbarone-treated mice [10,11,[30][31][32][33]. The striking difference between the numerous negative results and the latter has not been satisfactorily explained so far.…”

Chromosome analysis of mouse one-cell androgenones derived from a sperm nucleus exposed to topoisomerase II inhibitors at pre- and post-fertilization stages

“…Recently, however, micronucleus assays using human cultured somatic cells and mouse bone marrow cells have demonstrated that merbarone can induce both structural chromosome aberrations and aneuploidy [34,35]. Although the molecular mechanisms underlying the clastogenicity of merbarone 15 remain to be fully elucidated, the dysfunction of ooplasmic topo II by this agent may cause the torsional stress of DNA to accumulate in remodeled sperm chromatin, thereby generating DNA strand breaks.…”

Use of centromeric and telomeric DNA probes in in situ hybridization for differentiation of micronuclei induced by lomefloxacin

“…The negative control groups received PBS only, while the positive control groups were injected intraperitoneally with either 2 mg/kg mitomycin C (MMC; Sigma) or 2 mg/kg colchicine (COL; Sigma). The doses for the positive controls were chosen by reference to earlier in vivo studies [Schriever-Schwemmer and Adler, 1994;Schriever-Schwemmer et al, 1997;Attia et al, 2003;Attia, 2007]. The doses of lomefloxacin were selected on the basis of their effectiveness in inducing chromosomal alterations in mouse bone marrow [Singh et al, 2003].…”