A cytogenetic approach to evaluate in vivo somatic aneuploidy. Effects of diethylstilboestrol on mouse bone marrow cells

Abstract

A cytogenetic approach to estimate in vivo mitotic nondisjunction is proposed, based on chromosomal counting of mouse bone marrow metaphases differentially stained by bromodeoxyuridine (BrdU) incorporation. The method allows the simultaneous assessment of cell cycle delay, aneuploidy and SCE induction. Male mice, implanted with agar-coated BrdU tablets, were injected i.p. with diethylstilboestrol-diphosphate (DES-dp) in the dose range 10-300 mg/kg and killed 18 or 24 h later. To investigate possible sex differences a group of female mice of the same strain and age was injected with 100 mg/kg. As positive controls six males were injected with 1.8 mg/kg of vinblastine (VBL) sulphate. The induction of cell cycle delay was estimated by the relative frequency of first, second and third mitoses after treatment. In spite of a large biological variability, a dose-dependent delay of cellular proliferation kinetics was observed in DES-treated male mice. Treatment with VBL strongly delayed cell cycle progression, according to its antimitotic activity. Hyperploidy was assessed by chromosome counting of second generation metaphases only. After VBL injection, 10.2% of second mitoses were hyperploid, which is a frequency significantly higher than the 0.2% seen in control mice. No significant effect was detected at any DES dose. SCE induction was estimated in the same cells. A significant increase over the control frequency was observed after 200 and 300 mg/kg of DES. By analysis of variance (MANOVA) the dose--effect relationship was fitted by a quadratic model. A sex difference was observed only for spontaneous frequency of SCE with females showing higher levels probably due to their lower weight and relatively higher BrdU in vivo concentration.