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Cisplatin is a chemotherapeutic drug widely used in the treatment of several tumours, but this chemotherapy presents problems in terms of side-effects and patient resistance. The detection and determination of cisplatin-induced adducts and the relationship with the physiological or clinical effects of this drug under different repair conditions could be a good measure to assess patient's response to such chemotherapy. A new methodological approach to detect and quantify cisplatin adducts by use of high-performance liquid chromatography with inductively coupled plasma mass-spectrometric detection (HPLC-ICP-MS) and isotope-dilution analysis (IDA), is evaluated for its application in vivo, under different repair conditions. This analysis is combined with the use of the Comet assay, which detects DNA strand-breaks, and the w/w(+) SMART assay, which monitors induction of somatic mutation and recombination in Drosophila melanogaster in vivo under different conditions of nucleotide-excision repair. Results show that (i) cisplatin induces in Drosophila several adducts not detected in mammals. The two most abundant cisplatin-induced adducts, identified by electrospray-mass spectrometry as G monoadduct and G-G intrastrand cross-links, were quantified individually; (ii) cisplatin induces higher levels of G monoadducts and G-G cross-links in NER-proficient than in NER-deficient cells; (iii) the level of adducts correlates with their biological consequences, both in terms of DNA strand-breaks (tail-moment values), and of somatic mutation and recombination (frequency of mosaic eyes and clones in 10(4) cells), when the repair status is considered. This work demonstrates the validity and potential of the adduct detection and quantification methodology in vivo, and its use to correlate adducts with their genetic consequences., (© 2011 Elsevier B.V. All rights reserved.)

Cisplatin is one of the most effective chemotherapeutic agents, although its clinical use is limited by severe renal toxicity. This toxicity seems to be related to the accumulation of the drug in kidney tissues, leading to renal failure. For this reason, several compounds have been evaluated to ameliorate the nephrotoxicity induced by cisplatin. In the present investigation, we report the effect of the oral administration of selenomethionine before intraperitoneal cisplatin treatment. The preadministration of this Se species has been shown to have an important effect in reducing renal damage induced by cisplatin by increasing the excreted urea and improving creatinine clearance. Quantification of the level of DNA--cisplatin adducts in kidney and liver tissues was carried out by postcolumn isotope dilution analysis using liquid chromatography-inductively coupled plasma (LC-ICP-MS) as speciation set up. The level of DNA--cisplatin adducts in rats given Se-methionine in the drinking water before cisplatin administration was considerably lower in kidney tissues with respect to the animals drinking only water. Such effects were not observed in liver tissue. Initial speciation studies of Pt and Se conducted in kidney tissues of exposed animals by HPLC-ICP-MS have revealed the presence of cisplatin as part of a complex with Se-methionine, which can be eventually excreted into urine. This Pt--Se complex could explain the observed reduction of the kidney damage in Se-methionine-treated animals.

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.

The antitumoral effect of cisplatin [cis-diamminodichloroplatinum(II)] in mammals is related to its binding to DNA components. However, there is a lack of specific chemical methods to selectively detect those adducts formed in vivo at low concentrations. In this work, a new sensitive and selective method of determining cisplatin-DNA adducts based on the use of element-selective mass spectrometry is proposed, and the method is then applied to detect cisplatin adducts induced in vivo in somatic cells of Drosophila melanogaster. The bioanalytical strategy proposed here allows the determination of the most important DNA adduct formed between adjacent guanine units of the same DNA strand with cisplatin, and it is based on the coupling of capillary liquid chromatography (cap-LC) to inductively coupled plasma mass spectrometry (ICP-MS). This set-up allows the simultaneous monitoring of the Pt (from the drug) and P (from the DNA components) present in these adducts, once they have been cleaved by enzymatic hydrolysis of the DNA samples. Using this instrumental set-up, the adducts of cisplatin formed in vivo when D. melanogaster flies are exposed to different cisplatin concentrations can be detected and their concentration determined. The results obtained show a direct correlation between the concentration of cisplatin adducts, the induced genotoxic damage (measured as DNA strand breaks using the Comet assay) and the cisplatin concentration. [figure: see text] The work illustrates the complementary use of bioanalytical and biological information to study cisplatin interactions with DNA is vivo at biologically relevant concentrations of the drug.