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Please use this identifier to cite or link to this item: http://hdl.handle.net/10651/8748

Title: Quantitative profiling of in vivo generated cisplatin-DNA adducts using different isotope dilution strategies
Author(s): García Sar, Daniel
Montes Bayón, María
Blanco González, Elisa
Sierra Zapico, Luisa María
Aguado Ortiz, Leticia
Comendador García, Miguel Ángel
Köllensperger, Gunda
Hann, Stephan
Sanz Medel, Alfredo
Issue date: 2009
Publisher version: http://dx.doi.org/10.1021/ac901360f
Citation: Analytical Chemistry, 81(23), p. 9553-9560 (2009); doi:10.1021/ac901360f
Format extent: p. 9553-9560
Abstract: Platinum compounds are the major group of metal-based chemotherapeutic drug used in current practice and still a topic of intense investigation. The relative contribution of structurally defined cisplatin adducts with DNA to induce apoptosis and the cellular processing of these lesions is still poorly understood mostly due to the lack of sensitive and accurate analytical tools for in vivo studies. In this regard, two novel sensitive and selective strategies are proposed here to quantify cisplatin−DNA adducts generated in Drosophila melanogaster larvae and in head and neck squamous cell carcinoma cultures. The methods involve the isolation and enzymatic digestion of the DNA in the samples exposed to cisplatin and further quantification by high-performance liquid chromatography with inductively coupled plasma mass spectrometric detection (HPLC−ICPMS). Two different strategies, based on isotope dilution analysis (IDA), have been attempted and evaluated for quantification: species-unspecific (the postcolumn addition of a 194Pt-enriched solution) and the species-specific (by means of a synthesized isotopically enriched cisplatin (194Pt) adduct). For the second approach, the synthesis and characterization of the cisplatin adduct in a custom oligonucleotide containing the sequence (5′-TCCGGTCC-3′) was necessary. The adducted oligo was then added to the DNA samples either before or after enzymatic hydrolysis. The results obtained using these two strategies (mixing before and after enzymatic treatment) permit to address, quantitatively, the column recoveries as well as the efficiency of the enzymatic hydrolysis. Species-specific spiking before enzymatic digestion provided accurate and precise analytical results to clearly differentiate between Drosophila samples and carcinoma cell cultures exposed to different cisplatin concentrations.
URI: http://hdl.handle.net/10651/8748
ISSN: 0003-2700
Local identifier: 20090102
Sponsored: Financial support from the Ministry of Science and Innovation is acknowledged through the projects CTQ2007-60206/BQU and CTQ2006-02309
Project id.: MICINN/CTQ200760206/BQU
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