Induction of DNA crosslinks and DNA strand lesions by cyclophosphamide after activation by cytochrome P450 2B1

Abstract

Cyclophosphamide requires metabolic activation by cytochrome P450 to exert its genotoxic effects. Therefore in vitro studies on its mechanism of action have been limited to the use of self-activating derivatives of cyclophosphamide or to hepatocytes as an activating system. In this study we used a cell line of Chinese hamster lung fibroblasts (V79 cells), genetically engineered to express active cytochrome P450 2B1 as 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 alkaline elution assay. Compared to cyclophosphamide, its active metabolite 4-hydroperoxycyclophosphamide (4-OOH-CY) was about 250-fold more effective in induction of SL. Equimolar concentrations of phosphoramide mustard (50 μM), the ultimate DNA binding metabolite of cyclophosphamide, caused only about 50% of SL compared to 4-OOH-CY. A minimum of 12 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 μM). DNA crosslinks were observable after shorter incubation periods than single-strand breaks (6 h for cyclophosphamide and 1 h for 4-OOH-CY and phosphoramide mustard) and were no longer detectable at incubation periods of more than 20 h. Treatment of SD1 cells with ionizing radiation only, cyclophosphamide only, and radiation plus cyclophosphamide showed that SL induced by cyclophosphamide 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 observations give strong evidence that different types of SL were induced by cyclophosphamide and radiation. SD1 cells were able to repair the special kind of SL induced by radiation but not the SL caused by cyclophosphamide.