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A Cyclophosphamide/DNA Phosphoester Adduct Formed in Vitro and in Vivo¹

Grace Cancer Drug Center, Roswell Park Cancer Institute, Buffalo, New York 14263 [A. E. M., L. C., H. L. G.]; Southern Research Institute, Birmingham, Alabama 35255 [J. M. R.]; and Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, Alabama 35233 [D. H. H.]

The antitumor activity of cyclophosphamide is thought to be due to the alkylating activity of phosphoramide mustard, a metabolite of cyclophosphamide. Reaction of 2'-deoxyguanosine 3'-monophosphate and phosphoramide mustard resulted in the formation of several adducts that could be detected by high performance liquid chromatography (HPLC). One of these adducts, isolated and purified by HPLC, could be detected by ³²P postlabeling. This product was identified by UV, nuclear magnetic resonance, and mass spectrometry and by acid, base, and enzymatic hydrolysis to be 2'-deoxyguanosine 3'-monophosphate 2-(2-hydroxyethyl)aminoethyl ester. A combination of HPLC fractionation of digested DNA and ³²P postlabeling was used to detect this adduct in calf thymus DNA incubated in vitro with metabolically activated cyclophosphamide and in DNA from the liver of mice treated with cyclophosphamide. In these DNA samples the adduct occurred at a level of 1/10⁵ and 1/3 x 10⁷ nucleotides, respectively.

¹ This work was supported by NIH Grants CA-43567, BRSGS07RR-05648-23 and CA-24538. The NMR experiments were performed at the NMR Core Facility of the University of Alabama at Birmingham's Comprehensive Cancer Center which is supported by National Institutes of Health Grants RR03373 and CA13148 and by National Science Foundation Grant BBS-8611303. Mass spectroscopy was carried out by Dr. Richard Caprioli, University of Texas, Houston, TX, through arrangements made by Dr. G. B. Chheda of the Biophysics Department of Roswell Park Cancer Institute.

² To whom reprint requests should be addressed, at Roswell Park Cancer Institute, Grace Cancer Drug Center, Elm and Carlton Sts., Buffalo, NY 14263.

Received 8/13/90. Accepted 11/ 2/90.

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