Spontaneous and gamma-ray-induced sister chromatid exchanges in patients with carcinoma of cervix uteri.

The aim was to investigate whether there are differences in the spontaneous and gamma-ray-induced genomic instability in peripheral blood lymphocytes between untreated cervical cancer patients and healthy women using the sister chromatid exchange (SCE) assay as an indicator of chromosomal instability. Lymphocyte cultures from whole venous blood of 10 patients with cervical neoplasia and 10 healthy female volunteers were cultivated in vitro and irradiated using a 60Co-gamma source. Slides were prepared using the standard air-drying procedure and stained by the fluorescence-plus Giemsa (FPG) technique. The number of SCE and the number of chromosomes were assessed in second-division metaphases. A radiation dose-dependent increase of SCE/cell and SCE/chromosome values were found in healthy women as well as in patients, while statistical analysis has shown significantly higher SCE frequencies in healthy women as compared with patients. Cellular kinetics expressed as replication indices (RI) calculated from the frequency of cells in first cell division (M1), second cell division (M2) and third cell division (M3) were also significantly different, while observed RI were higher for patients than for control individuals. The results suggest that patients with carcinoma of the cervix uteri have chromosomal stability changes reflected in statistically different levels of spontaneous and induced SCE in comparison with healthy individuals. Despite the unknown mechanisms of SCE formation, it is felt that the changed SCE frequency, especially after mutagen treatment, may be used as a marker of increased cancer risk.

Chromosomal aberrations, sister chromatid exchanges and micronuclei induced by pentoxifylline in in vitro cultivated Chinese hamster cells (V79) and human blood lymphocytes.

Pentoxifylline (PTX) is a methylxanthine widely used in clinical practice. The mechanism of PTX effects on cellular and molecular level have not been fully explained yet. The present study was carried out to investigate the cytogenetic effect of this drug using cultured Chinese hamster V79 cells and human blood lymphocytes in vitro. The occurrence of chromosomal aberrations (CA), sister chromatid exchanges (SCE) and micronuclei (MN) was observed after the treatment of cells by different concentrations (0.002-2.0mg/ml) of PTX. In exposed V79 cells and lymphocytes as well, the dose-dependent increases of the above mentioned cytogenetic endpoints were found. The statistically significant increase has appeared at lower PTX concentrations in human lymphocytes than in V79 cells in all the investigated parameters. Our results show that, the applied concentrations of PTX has the clastogenic effect on in vitro cultured V79 cells and human lymphocytes. These findings are notable because of the frequent use of this drug and may serve as preliminary data to the further detailed examination of PTX action on molecular level.

Detection of MNNG-induced DNA lesions in mammalian cells; validation of comet assay against DNA unwinding technique, alkaline elution of DNA and chromosomal aberrations.

Human cells (VH10 or Hep G2) and hamster cells V79 were exposed to different concentrations of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and the level of DNA lesions was evaluated by the DNA unwinding technique, alkaline elution of DNA and the comet assay. All three methods were able to detect the effects of MNNG but with a clear difference in sensitivity. At low concentrations of MNNG the most sensitive method appeared to be the comet assay. After the short-term treatment the comet assay was able to detect the lesions induced by MNNG at approx. 0.1 microgram/ml, alkaline elution of DNA at 1 microgram/ml and DNA unwinding at 1-2 micrograms/ml. MNNG treated VH10 cells, human lymphocytes and V79 cells were also tested cytogenetically, confirming that MNNG induced chromosomal aberrations at concentrations > 1 microgram/ml in VH10 cells (short-term treatment): > 0.2 microgram/ml in V79 cells (long-term treatment) and > 8 micrograms/ml in human lymphocytes (long-term treatment). In some experiments we tried to increase the level of MNNG-induced DNA breaks with help of DNA repair inhibitors cytosine arabinoside (Ara C) and hydroxyurea (HU) which were applied either after or during MNNG treatment. Our results showed that the level of MNNG-induced lesions was increased by simultaneous treatment of cells with MNNG and Ara C and HU. 2 x 10(-5) M Ara C and 2 x 10(-3) MHU were as effective as 10-times higher concentrations of inhibitors. Ara C and HU increased the level of MNNG-induced DNA breaks mainly in combination with lower concentrations of MNNG (< 2 micrograms/ml). Rejoining of DNA breaks was observed in human cells VH10 and Hep G2 as well as in Chinese hamster cells V79 damaged by both lower and higher MNNG-concentrations. All methods showed that MNNG-induced DNA breaks had been gradually rejoined.