In vivo cytokinesis blocked micronucleus assay with carbendazim in rat fibroblasts and comparison with in vitro assays

A successful in vivo application of the cytokinesis blocked micronucleus as say for the detection of aneuploidy induced by carbendazim (CARB) was carri ed out in the granuloma pouch assay, This was performed in two ways: (i) in vivo exposure of the skin fibroblasts to cytochalasin B (cytB) and CARE, b y simultaneous injection of both substances into the pouch; (ii) in vivo ex posure to CARE followed by in vitro culturing of the fibroblasts in the pre sence of cytB, Only the first assay was successful. Injection of cytB ( wit h or without the test compound) into the pouch resulted in the induction of binucleate cells in vivo, up to a maximum of 5% at 1 mg cytB/pouch. After injection of CARE (0-50 or 0-10 mg/pouch) and cytB (1 mg) into the pouch, a neuploidy was determined in the isolated binucleate fibroblasts by fluoresc ence in situ hybridization with a general centromeric probe and combination s of chromosome-specific probes (19p + 19q, 4q + Yq), With all probes, the induction of chromosome loss and/or nondisjunction by CARE was very pronoun ced; at 10 mg CARE/pouch the total malsegregation frequency of chromosomes 4, 19 and Y was similar to 300/1000 binucleate cells, In an in vitro cytoki nesis block assay with CARE (0-2.5 mu g/ml) in primary skin fibroblasts the induced aneuploidy frequencies were as high as observed in the in vivo ass ay. The use of two probes for chromosome 19, which enabled the scoring of c hromosome breaks in addition to aneuploidy, revealed no significant inducti on of chromosome breaks by CARE. The frequency of polyploid mononucleate an d binucleate cells was decreased after CARE treatment, in both the in vivo and in vitro assays. However, in an additional in vitro assay without cytB a major induction of polyploidy from 2.5 mu g/ml CARE and above was observe d, showing that cytB may interfere with polyploidy induction.