Binding of Metabolites of Cyclophosphamide to DNA in a Rat Liver Microsomal System and in Vivo in Mice1

The stability of phosphoramide mustard, a metabolite of cyclophosphamide was studied at pH 7.2 and 37°Cusing 31Pnuclear magnetic resonance. The phosphorus signal of phosphoramide mustard disappeared with a half-life of 8 min indicating rapid conversion to other species. The final product, inorganic phos phate, appeared with a half-life of 105 min indicating that phos phoramide mustard was easily dephosphoramidated. A rat liver microsomal system was used to study the binding of [c/7/oroef/7y/-3H]cyclophosphamide to DNA. DNA was hydrolyzed in 0.1 N HCI:0.5 N NaCI at 80°Cfor 20 min, conditions known to convert phosphoramide mustard to nornitrogen mus tard with liberation of the phosphoramide residue. After such treatment three adducts were detected by high-performance liquid chromatography using several elution systems. They were all 7-substituted guanine adducts of nornitrogen mustard; two were monoalkylation products with an intact |A/-(2-chloroethyl)A/-[2-(7-guaninyl)ethyl]amine) or an hydroxylated mustard arm (A/-{2-hydroxyethyl)-A/-[2-(7-guaninyl)ethyl]aminei; the third adduct was a cross-linked product jA/,A/-bis[2-(7-guaninyl)ethyl]amine|. The relative abundance of these adducts depended on the length of the microsomal incubation. After 2 h, A/-(2-chloroethyl)-A/-[2-(guaninyl)ethyl]amine was the main product but after 6 h A/-(2-hydroxyethyl)-A/-[2-(7-guaninyl)ethyl]amine was most abundant, and at this time the cross-linked product represented 12% of the total adducts. The adducts in DNA depurinated readily and after 24 h at pH 7.0 and 37°C 70% of them had been liberated. The rate of depurination was decreased in the presence of 0.5 N NaCI. After short-term depurination in 0.1 N HCI at 25°Cthe primary alkylating species was phosphoramide mustard rather than nornitro gen mustard. In in vivo studies mice were given injections i.p. of 100 ¿¿Ci of Cyclophosphamide. Maximal levels of radioactivity had been incorporated into DNA between 2-7 h after injection; the specific activity of DNA from the kidney and lung exceeded that from the liver. While the level of radioactivity found in kidney DNA was rapidly reduced the rate of fall was lower in the lung. Between 24 and 72 h the specific activity of lung DNA exceeded that of kidney and liver DNA by a factor of 3:8. Lung is the prin cipal target tissue for tumor formation in mice after an i.p. in jection. 1Research sponsored by the National Cancer Institute, Department of Health and Human Services, under contract N01-CO-23909 with Litton Bionetics, Inc. The researchwas undertakenduring the tenure of an EleanorRooseveltFellowship of the InternationalUnionAgainst Cancer. 2Presentaddress: Instituteof OccupationalHealth,Haartmaninkatu1, SF-00290 Helsinki29, Finland.To whom requests for reprints should be addressed. Received1/22/85; revised 5/6/85; accepted 5/13/85. INTRODUCTION Few attempts have been made to characterize the DNA ad ducts produced by alkylating anticancer agents (1) even though their effect is thought to result from interaction with DNA (2). Cyclophosphamide presents a particular challenge since it acts in contrast to most other alkylating agents used after metabolic activation (3-5). Cyclophosphamide is initially oxidized by the hepatic cytochrome P-450-dependent mixed-function oxidases into 4-hydroxycyclophosphamide, which is thought to exist in equilibrium with its ring-opened tautomer, aldophosphamide. These are converted nonenzymatically or metabolized by alde hyde dehydrogenase and aldehyde oxidase to the relatively nontoxic metabolites 4-ketophosphamide, carboxyphosphamide, and aldophosphamide. Alternatively they can spontaneously form the cytotoxic species acrolein and phosphoramide mustard (6-9). Phosphoramide mustard undergoes cleavage of the phos phoramide residue to form NOR3 (8, 10), a metabolite of Cyclo phosphamide which has been detected in human urine (11). The structures of Cyclophosphamide, phosphoramide mustard, and nornitrogen mustard are shown in Chart 1. Model studies have been carried out in vitro to characterize the reaction products of phosphoramide mustard with nucleosides and nucleotides (10,12-16). Particularly the work of Mehta et al. (12), Mehta and Ludlum (13), and Vu ef al. (10) has helped to understand the reactions of PAM with nucleic acid constitu ents. The preferred site of reaction is the N-7 position of guanine and a considerable labilization of nucleoside products has been noted (12, 14-16). Cyclophosphamide can generate alkylating species capable of binding to DNA (17,18) and poly nucleotides (13) in the presence of a microsomal system. The alkyl moiety was thought to be phosphoramide mustard (13). In another study using a microsomal system, binding to nucleic acids was the result of reaction with phosphoramide mustard, whereas with acrolein (19) most of the binding was to protein. The present work was undertaken to characterize the DNA binding products of Cyclophosphamide in a rat liver microsomal system. In vivo studies were also carried out to determine the level of binding in different tissues and to see if the level of binding could be shown to be related to the organotropic effects of this compound in animals. MATERIALS AND METHODS Preparation of Substituted-Guanine Standards. Nornitrogen mustard-guanine standards were prepared by heating at reflux overnight a 3The abbreviationsused are: NOR, nornitrogen mustard; PAM, phosphoramide mustard; NOR-G, N-(2-chtoroethyl)-N-[2-(7-guaninyl)ethyl]amine; NOR-G-OH, W-(2-hydroxyethyl)-N-[2-<7-guaninyl)ethyl]amine; G-NOR-G, W,W-bis[2-<7-guaninyl)ethyl]amine; PAM-G, W-(2-chloroethyl)-W-[2-(7-guaninyl)ethyl]phosphorodiamic acid; HPLC, high performance liquid chromatography; NMR, nuclear magnetic resonance;HEPES,4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid. CANCER RESEARCH VOL. 45 SEPTEMBER 1985