Interaction of cyclophosphamide with DNA in isolated rat liver cell nuclei

Isolated rat liver cell nuclei were incubated with the alkylating cytostatic drug cyclophosphamide (CPA) in the presence of a microsomal activation system. Digestion of the 3H-CPA-treated nuclei with DNase I and micrococcal nuclease, respectively, showed that the CPA-modified DNA apparently has become resistant against such enzymatic attack. For analysis of the DNA-CPA reaction products, the DNA was isolated under mild conditions and degraded enzymatically. In cell nuclei whose DNA had been prelabeled with 32P in vivo, a predominant binding of 3H-CPA (about 50%) to the DNA phosphate groups was observed. Terminal phosphate groups apparently play an important role in this reaction. Neutral heating of DNA from 3H-CPA-treated nuclei liberated up to 60% of the initially bound 3H-radioactivity. The results are discussed in relation to the recent data concerning the route of decomposition of activated CPA to phosphoramide mustard and acrolein.