No increase in radiation-induced chromosome aberration complexity detected by m-FISH after culture in the presence of 5’-bromodeoxyuridine

The thymidine analogue, 5’-bromodeoxyuridine (BrdU), is a known mutagen that is routinely introduced into culture media for subsequent Harlequin stain analysis and determination of cell cycle status. Previously, we examined the induction of chromosome aberrations in human peripheral blood lymphocytes (PBL) known to be in their 1st cell division following exposure to a low dose (0.5 Gy, average one -particle per cell) of high-LET α-particles. We found complex chromosome aberrations to be characteristic of exposure to high-LET radiation and suggested the features of complex exchange to reflect qualitatively the spatial deposition of this densely ionising radiation. To exclude the possibility that BrdU addition post-irradiation influenced the complexity of chromosomal damage observed by m-FISH, the effect of increasing BrdU concentration on aberration complexity was investigated. Comparisons between BrdU concentration (0, 10, and 40 M) and between sham- and α-particle irradiated PBL, were made both independently and in combination to enable discrimination between BrdU and high-LET radiation effects. Aberration type, size, complexity and completeness were assessed by m-FISH, and the relative progression through cell division was evaluated. We found no evidence of any qualitative difference in the complexity of damage as visualized by m-FISH but did observe an increase in the frequency of complex exchanges with increasing BrdU concentration indicative of altered cell cycle kinetics. The parameters measured here are consistent with findings from previous in vitro and in vivo work, indicating that each complex aberration visualised by m-FISH is characteristic of the structure of the high-LET α-particle track and the geometry of cell irradiated

Dependence of the frequency of harlequin-stained cells on BrdU concentration in human lymphocyte cultures.

After exposure of stimulated human lymphocytes to 5-bromodeoxyuridine (BrdU), metaphase cells that have replicated for 1, 2 or 3 cycles can be unequivocally identified with the FPG-staining technique (fluorescence plus Giemsa). The application of this method can remove a source of error in dose-response data, especially when it is used for cytogenetic dose estimation after exposure to radiation. Such estimates can be misleading when the chromosome damage is not analysed at the first post-irradiation mitosis because some aberration types are lost at cell division or the cells die owing to irregular mitosis. In mutagenicity testing we deal with the special problem of simultaneous treatment of cells with the test substance and BrdU. Assuming BrdU cannot be applied owing to a synergistic clastogenic effect, conventionally stained cells must be analysed. Depending on the frequency of second-division cells, the aberration frequency will be more or less underestimated. Thus, for a meaningful correction of this error, the ratio of first- and second-division cells must be determined in parallel cultures with BrdU. Nevertheless, in this case, in addition, a synergistic or related effect, between BrdU and the clastogen, on cell kinetics must be excluded. Because BrdU itself has an inhibitory influence on the cell proliferation, we first tried to find concentrations that do not induce such an effect. We present here results from preliminary experiments on the dependence of the frequency of second-division cells on BrdU concentrations. With increasing concentrations of BrdU, there is an exponential decline of the frequency of harlequin-stained metaphases with different slopes for the different donors. One can assume that with concentrations of the plateau up to 10-5 M, BrdU has no inhibitory effect on the cell proliferation. With increasing concentrations, cells are delayed in proceeding through the next cell cycle, i.e. within a culture time of 48 hr, non-harlequin-stained metaphases (first division) begin to predominate

Azathioprine, a clastogen in human somatic cells? Analysis of chromosome damage and SCE in lymphocytes after exposure <em>in vivo </em>and <em>in vitro</em>.

Chromosomal analyses in lymphocytes of 28 patients with multiple sclerosis were carried out before, during and after Azathioprine (Aza) therapy. Only a higher incidence of gaps was found in treated patients than in a group of healthy persons but not in comparison with untreated patients. Similarly, no significant clastogenic effect was observed in vitro after short-term and long-term treatment of unstimulated and stimulated lymphocytes with concentrations of 1-100 &mu;g Aza per ml. Treatment of cultures with 0.0001-4.0 &mu;g/ml did not yield increased SCE frequencies. The absence of any significant clastogenic effect of therapeutic doses of Aza on human somatic cells is deduced from an evaluation of previously published data and from the present results

Chromosome changes in lymphocytes after occupational exposure to toluene.

Cytogenetic analyses were carried out in peripheral lymphocytes from 20 male workers exposed only to toluene in a rotogravure plant for more than 16 years. As compared with a group of 24 unexposed controls, significantly higher yields of chromatid breaks, chromatid exchanges and gaps were observed. The number of SCEs was significantly increased in smoking and non-smoking toluene-exposed workers compared with the corresponding control groups