N-(2-hydroxyethyl)-N-[2-(7-guaninyl)ethyl]amine, the putative major DNA adduct of cyclophosphamide in vitro and in vivo in the rat.
The anti-cancer agent, cyclophosphamide, metabolises to the cytotoxic alkylating agent phosphoramide mustard, which can be dephosphoramidated to give nornitrogen mustard. A rat liver mitochondrial supernatant system was used to study the binding of [chloroethyl 3H]cyclophosphamide to DNA. The reacted DNA was acid-hydrolysed and one major adduct was identified using Sephadex G-10 chromatography, followed by HPLC, using reversed-phase or ion-exchange systems. Further studies, using [14C]guanine as reaction substrate for [chloroethyl 3H]cyclophosphamide, phosphoramide mustard or nornitrogen mustard, demonstrated the main adduct from each reaction had identical chromatographic properties in these systems. The radiolabelled ratio in the [3H]cyclophosphamide-[14C]guanine reaction demonstrated a monoadducted product. From this evidence and from 1H NMR data, the common adduct was putatively identified as a hydroxylated nornitrogen mustard adduct (N-(2-hydroxyethyl)-N-[2-(7-guaninyl)ethyl]amine). In in vivo studies, rats were injected intraperitoneally with 2.775 MBq [3H]cyclophosphamide. Total organ [3H] content and DNA binding levels were ascertained. Maximal levels of [3H] binding to DNA were seen between 1-4 hr with the highest binding levels observed in the bladder. The in vivo adduct was shown, using various HPLC systems, to co-chromatograph with the in vitro adduct and thus the main in vivo adduct was putatively identified as N-(2-hydroxyethyl)-N-[2-(7-guaninyl)ethyl]amine.
Benson
A J
AJ
Cancer Research Unit, University of York, Heslington, U.K.
Martin
C N
CN
Garner
R C
RC
D000478:Alkylation; D000818:Animals; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003520:Cyclophosphamide / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D006147:Guanine / Q000031:analogs & derivatives* / Q000302:isolation & purification / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007274:Injections, Intraperitoneal; D007668:Kidney / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D051381:Rats; D013056:Spectrophotometry, Ultraviolet; D001743:Urinary Bladder / Q000378:metabolism
D006147:Guanine; D003520:Cyclophosphamide; D004247:DNA; C056546:N-(2-hydroxyethyl)-N-(2-(7-guaninyl)ethyl)amine