Evaluation of thresholds for benomyl- and carbendazim-induced aneuploidy in cultured human lymphocytes using fluorescence in situ hybridization

Abstract

Threshold mechanisms of activity for mutagenic agents have been debated for some time, especially for those substances which induce aneuploidy by inhibiting mitotic spindle function. No observed effect levels (NOELs) or “practical thresholds” have been demonstrated for several aneugens both in vitro and in vivo generally by either counting chromosomes in metaphase preparations or by observing micronuclei. Recently, fluorescence in situ hybridization (FISH) has proven to be a sensitive and useful technique for the assessment of aneuploidy at low concentrations. Using binucleate human lymphocytes coupled with FISH, we have been able to characterize a threshold mechanism of action for two spindle inhibitors, benomyl and its active metabolite, carbendazim. Test chemicals were added 24 h following culture initiation. After a further 20 h, cytochalasin B was added, and cells were harvested 28 h later (72 h post initiation). The distribution of chromosomes between the nuclei of binucleate cells was evaluated by fluorescence microscopy for the simultaneous detection of centromeres labeled with FITC (green) or Cy-3 (red). Six human chromosomes were investigated in pairs (1 and 8, 11 and 18, and X and 17). Abnormalities were classified as chromosome loss (including centromeric positive micronuclei), chromosome gain, non-disjunction, or polyploidy. Dose–response data were generated over a range of closely spaced concentrations at 100 ng/ml intervals. The threshold, defined as the lowest “effect” concentration using statistical methods, was determined for each chromosome. Non-disjunction proved to be the most sensitive endpoint for the detection of aneuploidy occurring at higher frequencies and lower concentrations. Results for the six chromosomes demonstrated similar dose–response data which included a series of concentrations with no statistically significant increase above background, followed by a second range of higher concentrations with a statistically significant, concentration-dependent increase. Nearly equimolar threshold concentrations were determined for benomyl- and carbendazim-induced non-disjunction.