Chlorambucil-adducts in DNA analyzed at the oligonucleotide level using HPLC-ESI MS.
Chlorambucil (N,N-bis(2-chloroethyl)-p-aminophenylbutyric acid) is a bifunctional alkylating drug belonging to the nitrogen mustard group and is widely used as an anticancer agent. As the antitumor activity of the nitrogen mustards is based on the formation of adducts with genomic DNA, calf thymus DNA-Chlorambucil adducts were the major target in this study. Calf thymus DNA was incubated with Chlorambucil to induce the formation of a wide variety of adducts. Subsequently, enzymatic digestion of the DNA was performed using Benzonase and Nuclease S1 aiming at the production of oligonucleotides. Separation and structure elucidation of the individual DNA-Chlorambucil adducts was achieved using HPLC interfaced to electrospray ionization ion trap mass spectrometry. Both trinucleotide and tetranucleotide Chlorambucil adducts were detected. The majority of the detected trinucleotide adducts involved monofunctional alkylation with guanine being the hotspot for alkylation. Only a few bifunctional trinucleotide adducts both intra- and interstrand cross-links were found. On the contrary, cross-linked adducts were the major detected tetranucleotides in which the intrastrand cross-links predominated over the interstrand cross-links. To a lesser extent, monofunctional guanine alkylated tetranucleotides were detected as well. With MS(n) experiments, the detailed structures of Chlorambucil adducts of the tri- and tetranucleotides were determined.
Mohamed
Dalia
D
Humboldt-Universitaet zu Berlin, Department of Chemistry, Applied Analytical and Environmental Chemistry, Brook-Taylor-Strasse 2, 12489 Berlin, Germany.
Mowaka
Shereen
S
Thomale
Jürgen
J
Linscheid
Michael W
MW
D000478:Alkylation; D000818:Animals; D000970:Antineoplastic Agents / Q000737:chemistry; D018906:Antineoplastic Agents, Alkylating / Q000737:chemistry; D002699:Chlorambucil / Q000737:chemistry* / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003432:Cross-Linking Reagents; D004247:DNA / Q000032:analysis / Q000737:chemistry*; D018736:DNA Adducts / Q000032:analysis*; D004249:DNA Damage / Q000187:drug effects; D006147:Guanine / Q000737:chemistry; D007202:Indicators and Reagents / Q000494:pharmacology*; D051379:Mice; D009588:Nitrogen Mustard Compounds / Q000737:chemistry; D009841:Oligonucleotides / Q000032:analysis / Q000737:chemistry*; D053719:Tandem Mass Spectrometry
D000970:Antineoplastic Agents; D018906:Antineoplastic Agents, Alkylating; D003432:Cross-Linking Reagents; D018736:DNA Adducts; D007202:Indicators and Reagents; D009588:Nitrogen Mustard Compounds; D009841:Oligonucleotides; D002699:Chlorambucil; D006147:Guanine; D004247:DNA; C427203:calf thymus DNA