Measurement of DNA cross-linking in patients on ifosfamide therapy using the single cell gel electrophoresis (comet) assay

The single cell gel electrophoresis comet assay has become established as a sensitive technique for measuring DNA strand breaks. The technique has bee n modified to allow the sensitive detection and quantitation of DNA interst rand cross-linking at the single cell level. Cells are irradiated immediate ly before analysis to deliver a fixed level of random strand breakage. Afte r embedding of cells in agarose and lysis, the presence of cross-links reta rds the electrophoretic mobility of the alkaline denatured cellular DNA, Cr oss-links are, therefore, quantitated as the decrease in the comet tail mom ent compared with irradiated controls, Using this method, a linear response of cross-linking versus dose of chlorambucil over a wide dose range was de monstrated in human lymphocytes after drug treatment ex vivo. The method wa s also sensitive enough to determine cross-linking in clinical samples afte r chemotherapy, For example, crosslinking was observed in the lymphocytes o f patients receiving ifosfamide (3 g/m(2)/day) as a continuous infusion for 3-5 days or as a 3-h infusion daily for 3 days. Cross-links were detected in all patients within 3 h, with no evidence of DNA single strand break for mation. In patients receiving continuous infusion, a plateau of cross-linki ng was reached by 24 h, In the patients receiving ifosfamide over 3 h, a cl ear decrease in the peak level of cross-linking was observed before subsequ ent infusions.