INDUCTION OF DNA CROSS-LINKS AND DNA STRAND LESIONS BY CYCLOPHOSPHAMIDE AFTER ACTIVATION BY CYTOCHROME-P450 2B1

Cyclophosphamide requires metabolic activation by cytochrome P450 to e xert its genotoxic effects. Therefore in vitro studies on its mechanis m of action have been limited to the use of self-activating derivative s of cyclophosphamide or to hepatocytes as an activating system. In th is study we used a cell line of Chinese hamster lung fibroblasts (V79 cells), genetically engineered to express active cytochrome P450 2B1 a s the sole observable cytochrome P450 (SD1 cells). An increase in DNA strand lesions (SL: DNA single-strand breaks and alkali labile sites) was observed between 0.5 and 1.5 mM cyclophosphamide (24 h incubation) which could be classified as alkali labile sites using a modified alk aline elution assay. Compared to cyclophosphamide, its active metaboli te 4-hydroperoxycyclophosphamide (4-OOH-CY) was about 250-fold more ef fective in induction of SL. Equimolar concentrations of phosphoramide mustard (50 mu M), the ultimate DNA binding metabolite of cyclophospha mide, caused only about 50% of SL compared to 4-OOH-CY. A minimum of 1 2 h of incubation of SD1 cells was needed for cyclophosphamide (1 mM) until SL were detectable, compared to only 2 h for 4-OOH-CY and 1.5 h for phosphoramide mustard (50 mu M). DNA crosslinks were observable af ter shorter incubation periods than single-strand breaks (6 h for cycl ophosphamide and 1 h for 4-OOH-CY and phosphoramide mustard) and were no longer detectable at incubation periods of more than 20 h. Treatmen t of SD1 cells with ionizing radiation only, cyclophosphamide only, an d radiation plus cyclophosphamide showed that SL induced by cyclophosp hamide were not repaired during incubation with fresh culture medium ( 24 h). However, an efficient repair of SL caused by ionizing radiation was observed and was not inhibited by cyclophosphamide. These observa tions give strong evidence that different types of SL were induced by cyclophosphamide and radiation. SD1 cells were able to repair the spec ial kind of SL induced by radiation but not the SL caused by cyclophos phamide.