Analysis of aclarubicin-induced cell death in human fibroblasts.

In the present study we investigated the mode of cell death induced by aclarubicin (ACL) in trisomic (BB) and normal (S-2) human fibroblasts. Cells were incubated with ACL for 2h and then cultured in drug-free medium for up to 96h. Using fluorescence microscopy, agarose gel electrophoresis and comet assay we demonstrate that ACL induced time-dependent morphological and biochemical changes in both cell types. The population of apoptotic cells, analysed by acridine orange and ethidium bromide nuclear staining reached its maximum at 24-48h. Prolonged post-treatment time progressively increased the level of necrotic cells. At 24-48h time points we also observed a significant increase in caspase-3 activity, oligonucleosomal DNA fragmentation and DNA strand breaks. Cotreatment of cells with the specific caspase-3 inhibitor Ac-DEVD-CHO partly reduced the extent of apoptosis and necrosis and DNA degradation. In conclusion, trisomic and normal fibroblasts demonstrate similar response to aclarubicin treatment. Drug induced the apoptotic and necrotic pathway of cell death that was mediated by caspase-3.

Comparison between the genotoxicity of cis-Pt(ll) complex of 3-aminoflavone and cis-DDP in lymphocytes evaluated by the comet assay.

cis-bis(3-aminoflavone)dichloroplatinum(II) [cis-Pt(II) complex of 3-aminoflavone] is an analog of cis-DDP characterized by low cytotoxicity and anticancer properties in vivo. In order to evaluate genotoxic properties of this chemical compound, the comet assay in human lymphocytes was used. The analysis of DNA damage after 1-h cell incubation with cis-Pt(II) complex of 3-aminoflavone and cis-DDP was carried out, and DNA repair kinetics were evaluated after 0.5-h, 1-h, and 1.5-h postexposure incubation. It has been shown that cis-Pt(II) complex of 3-aminoflavone causes the increase in tail moments in comparison with cis-DDP. On the other hand, the decrease in these values caused by cis-DDP was connected mainly with the presence of DNA and DNA-protein crosslinks. The decrease in tail moments after cis-Pt(II) complex of 3-aminoflavone lymphocyte treatment was already observed after 0.5-h postexposure incubation, whereas in the variant with hydrogen peroxide application these values after cis-Pt(II) complex of 3-aminoflavone addition were higher in comparison with cis-DDP. Results obtained on the basis of the comet assay could confirm the occurrence of DNA breaks and cross-links induced by cis-Pt(II) complex of 3-aminoflavone.

Induction of chromosome aberrations in cultured human lymphocytes treated with ethoxyquin.

The chromosomal aberration test was employed to investigate the effect in vitro of a known antioxidant and food preservative, ethoxyquin (EQ, 1,2-dihydro-6-ethoxy-2,2,4-trimethylquinoline) on human chromosomes. The studies were undertaken because there are no published in vitro data on genotoxicity of EQ in mammalian cells and there are many reports pointing out that it may be harmful to animals and human beings. Lymphocytes obtained from three healthy donors were incubated with EQ (0.01-0.5mM) both with and without metabolic activation. Stability studies performed by HPLC analysis showed that EQ was stable under the conditions of the lymphocyte cultures. The results of the chromosome aberration assay showed that EQ induces chromosome aberrations: gaps and breaks as well as dicentrics and atypical translocation chromosomes.

Vicia faba chromosomal aberration assay.

A collaborative study involving laboratories in six countries was initiated under the sponsorship of the International Programme on Chemical Safety (IPCS) to determine the sensitivity, efficiency and reliability of the Vicia faba root tip meristem chromosomal aberration assay using a standardized protocol. The six laboratories that participated in this study were located in the Slovak Republic, India, Japan, Poland, Sweden and the USA. All laboratories adhered to a standardized protocol for the Vicia faba chromosomal aberration assay. Four coded chemicals, azidoglycerol (AG), N-methyl-N-nitrosourea (MNU), sodium azide (NaN3) and maleic hydrazide (MH) were tested with the Vicia faba chromosomal aberration assay. Of the four chemicals, three (MH, AG and MNU) were found to be clastogenic and gave a concentration related response. However, the results of NaN3 were equivocal which might be explained by the stability of NaN3. The conclusions from this study suggest that the Vicia faba chromosomal aberration bioassay is an efficient and reliable short-term bioassay for the rapid screening of chemicals for clastogenicity.

The effect of herbicide "Chwastox plynny 30" on the cell cycle parameters in root meristem of Vicia faba L. subsp. minor.

The duration of the cell cycle and its phases after the treatment with herbicide "Chwastox plynny 30" was calculated using 3H-thymidine labelling method. Inhibition of DNA synthesis and marked prolongation of G2 + 1/2 M phase were observed. Tested herbicide caused a significant lowering in the mitotic activity and accumulation of metaphase cells.

The lack of correlation between the rate of rRNA transport from nucleoli into cytoplasm and the duration of G1 and G2 phases in root meristem cells.

In root meristems of 3 species (Secale cereale L., Vicia faba L. subsp. minor, Allium cepa L.) the durations of cell cycles and their phases were calculated using 3H-thymidine labelling. In the above species and in Helianthus annuus L. (parameters of the cell cycle determined earlier) the G1 and G2 phase durations were different: G1 + 1/2 M from 3 h to 6.1 h, G2 + 1/2 M from 1.1. h to 8.3 h, depending on the species. The rate of rRNA transport from nucleoli into cytoplasm during recovery after cold treatment was calculated from our data presented earlier. The results indicate that in 4 species studied there is no correlation (at P = 0.05) between the rate of rRNA transport and the duration of G1 and G2 phases.