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Position-specific trapping of topoisomerase II by benzo[a]pyrene diol epoxide adducts: implications for interactions with intercalating anticancer agents.

ABSTRACT: DNA topoisomerase II (Top2) is the target of some of the most effective anticancer DNA intercalators. To determine the effect of intercalating ligands at defined positions relative to a known DNA cleavage site for human Top2alpha, we synthesized oligodeoxynucleotides containing single trans-opened benzo[a]pyrene 7,8-diol 9,10-epoxide (DE) deoxyadenosine (dA) adducts of known absolute configuration, placed at specific positions in a duplex sequence containing staggered Top2 cleavage sites on both strands. Because the orientations of the intercalated hydrocarbon are known from NMR solution structures of duplex oligonucleotides containing these dA adducts, a detailed analysis of the relationship between the position of intercalation and trapping of Top2 is possible. Our findings demonstrate that (i) Top2 cleavage complexes are trapped by intercalation of the hydrocarbon at either of the staggered cleavage sites or immediately adjacent to the base pairs flanking the cleavage sites within the stagger; (ii) both concerted and nonconcerted cleavage by both subunits of a Top2 homodimer were detected depending on the position of the benzo[a]pyrene DE dA adduct; and (iii) intercalation immediately outside of the staggered Top2 cleavage site, and to a lesser extent in the middle of the stagger, prevents Top2 from cleaving DNA at this site, consistent with the effect of some intercalators as suppressors of Top2-mediated DNA cleavage. These results identify specific binding sites for intercalators that result in trapping of Top2. Such poisoning of Top2 by bulky polycyclic aromatic hydrocarbon DE adducts constitutes a potential mechanism for their carcinogenic activity.

SUBMITTER: Khan QA

PROVIDER: S-EPMC218786 | biostudies-literature | 2003 Oct

REPOSITORIES: biostudies-literature

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Position-specific trapping of topoisomerase II by benzo[a]pyrene diol epoxide adducts: implications for interactions with intercalating anticancer agents.

Khan Qasim A QA Kohlhagen Glenda G Marshall Richard R Austin Caroline A CA Kalena Govind P GP Kroth Heiko H Sayer Jane M JM Jerina Donald M DM Pommier Yves Y

Proceedings of the National Academy of Sciences of the United States of America 20031001 21

DNA topoisomerase II (Top2) is the target of some of the most effective anticancer DNA intercalators. To determine the effect of intercalating ligands at defined positions relative to a known DNA cleavage site for human Top2alpha, we synthesized oligodeoxynucleotides containing single trans-opened benzo[a]pyrene 7,8-diol 9,10-epoxide (DE) deoxyadenosine (dA) adducts of known absolute configuration, placed at specific positions in a duplex sequence containing staggered Top2 cleavage sites on both ...[more]

PMID: 14523238

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Project description:Electrophilic diol epoxide metabolites are involved in the carcinogenicity of benzo[a]pyrene, one of the widely studied polycyclic aromatic hydrocarbons (PAHs). The exposure of humans to this PAH can be assessed by measuring stable blood protein adducts, such as to histidine and lysine in serum albumin, from their reactive metabolites. In this respect, measurement of the adducts originating from the genotoxic (+)-anti-benzo[a]pyrene diol epoxide is of interest. However, these are difficult to measure at such low levels as are expected in humans generally exposed to benzo[a]pyrene from air pollution and the diet. The analytical methods detecting PAH-biomarkers still suffer from low selectivity and/or detectability to enable generation of data for calculation of in vivo doses of specific stereoisomers, for evaluation of risk factors and assessing risk from exposures to PAH. Here, we suggest an analytical methodology based on high-pressure liquid chromatography (HPLC) coupled to high-resolution tandem mass spectrometry (MS) to lower the detection limits as well as to increase the selectivity with improvements in both chromatographic separation and mass determination. Method development was performed using serum albumin alkylated in vitro by benzo[a]pyrene diol epoxide isomers. The (+)-anti-benzo[a]pyrene diol epoxide adducts could be chromatographically resolved by using an HPLC column with a pentafluorophenyl stationary phase. Interferences were further diminished by the high mass accuracy and resolving power of Orbitrap MS. The achieved method detection limit for the (+)-anti-benzo[a]pyrene diol epoxide adduct to histidine was approximately 4 amol/mg serum albumin. This adduct as well as the adducts to histidine from (-)-anti- and (+/-)-syn-benzo[a]pyrene diol epoxide were quantified in the samples from benzo[a]pyrene-exposed mice. Corresponding adducts to lysine were also quantified. In human serum albumin, the anti-benzo[a]pyrene diol epoxide adducts to histidine were detected in only two out of twelve samples and at a level of approximately 0.1 fmol/mg.

Dataset Information

Position-specific trapping of topoisomerase II by benzo[a]pyrene diol epoxide adducts: implications for interactions with intercalating anticancer agents.

Publications

Position-specific trapping of topoisomerase II by benzo[a]pyrene diol epoxide adducts: implications for interactions with intercalating anticancer agents.

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