Effects of ethidium bromide on DNA loop organisation in human lymphocytes measured by anomalous viscosity time dependence and single cell gel electrophoresis

The effects of ethidium bromide (EtBr) on human lymphocytes were studied by the method of anomalous viscosity time dependence (AVTD) and by the comet assay. EtBr at low concentrations increased the maximum viscosity and time of radial migration as measured with AVTD at neutral conditions of lysis. A pronounced relaxation of DNA loops was observed with the neutral comet ass ay. The maximal comet length corresponded to 2 Mb DNA loops. At high concen trations of EtBr, 2. mg/ml, significant reduction in AVTD below control lev el was seen that suggested hypercondensation of chromatin. The hypercondens ation was directly observed with the neutral comet assay. EtBr did not indu ce DNA strand breaks as measured by the alkaline comet assay. The hypercond ensed nuclei could be decondensed by irradiation with gamma-rays or exposur e to light. The data provide evidence that EtBr at high concentrations resu lted in hypercondensation of chromatin below control level. The comet assay confirmed that the increase in AVTD peaks deals with relaxation of loops a nd AVTD decrease is caused by chromatin condensation. The prediction of the AVTD theory for a correlation between time of radial migration and condens ation of chromatin was verified. Further, the data show that the comet assa y at neutral conditions of lysis is rather sensitive to DNA loop relaxation in the absence of DNA damage. Finally, donor specificity was found for the hypercondensation. (C) 1999 Elsevier Science B.V. All rights reserved.