Chlorambucil-Adducts in DNA Analyzed at the Oligonucleotide Level Using HPLC-ESI MS

Abstract

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.

Characterization of Nitrogen Mustard Formamidopyrimidine Adduct Formation of Bis(2-chloroethyl)ethylamine with Calf Thymus DNA and a Human Mammary Cancer Cell Line

“…The chemistry and reactivity of nitrogen 8-10, 18, 20, 24, 47-56 and sulfur 44, 57-64 mustards with nucleosides and DNA in vitro is well studied. There are also numerous studies that examine the mustard derived DNA adducts from treated cell culture and animals, 46,48,50,54,56,59,64,65 including white blood cells of patients on chemotherapeutic regimens that include a nitrogen mustard.…”

“…53, 57 A variety of methods have been used to identify these adducts including 32 P-postlabeling, 47, 58 co-chromatography by HPLC with authentic standards, 8, 9, 24, 48, 57 and mass spectrometry. 18, 20, 49-52, 56, 59, 64 Recently, tandem mass spectrometry with stable isotope dilution has been employed to identify and quantitate some of these DNA adducts. 53, 54, 61-63 Collectively, the studies indicate that these electrophiles react predominantly at the N7 atom of dG, although N3-dA modification can also be significant.…”

Targeted gene correction using psoralen, chlorambucil and camptothecin conjugates of triplex forming peptide nucleic acid (PNA)

“…Psoralen can form DNA interstrand cross-links upon activation with UVA radiation, and the repair of such cross-links may involve DNA double strand breaks and/or recombination. [12][13][14] Chlorambucil readily forms mono-adducts (as well as some crosslinks) with dsDNA, 15 whereas camptothecin does not react with the DNA strand directly, but rather interfere with topoisomerase I activity by blocking the re-sealing of the DNA, which may eventually result in frank strand breaks. 16 Surprisingly, we find that sequence-targeted PNAs that can react covalently with the DNA helix exhibit a highly inhibitory effect on gene correction in nuclear extract and in a cell culture, extra-chromosomal assay.…”

Toward Hypoxia-Selective DNA-Alkylating Agents Built by Grafting Nitrogen Mustards onto the Bioreductively Activated, Hypoxia-Selective DNA-Oxidizing Agent 3-Amino-1,2,4-benzotriazine 1,4-Dioxide (Tirapazamine)

“…3,5−12 The predominant site of DNA alkylation by nitrogen mustards is the N7-atom of guanine residues. 6,8,9 …”