The effect of prolonged incubations and heat denaturation on melphalan-induced DNA cross-links as measured by the ethidium bromide fluorescence assay

Abstract The ethidium bromide fluorescence assay detects DNA interstrand crosslinks following heat denaturation of DNA on the basis of a 20–25 fold enhancement of ethidium bromide fluorescence in the presence of double stranded DNA. This assay has been utilized to detect DNA cross-links produced by melphalan in lymphocytes from chronic lymphocytic leukemia patients. The percentage of DNA cross-links (C t ) in these cells did not vary linearly with the concentration of melphalan, possibly as a result of DNA fragmentation during a 16 h lysis to degrade RNA. In order to investigate this, DNA was exposed to melphalan and then the C t was determined immediately after maximal DNA cross-link formation or after a 16 h incubation. The additional incubation period did not alter the linear relationship between C t and melphalan concentration. Further, the DNA cross-links produced by melphalan does not appear to be heat labile since varying the heat denaturation period from 5 to 15 min had no effect on C t . These results suggest that this assay can accurately quantitate the percentage of DNA cross-links produced by melphalan and that the curvilinear relationship between C t and melphalan concentration in malignant human lymphocytes is not due to the ethidium bromide fluorescence assay.