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Cisplatin has been extensively studied as an antitumor agent since the late 1960s. However the mode of action for the efficacy and adverse effects of cisplatin are poorly understood. It was previously believed that the cisplatin1,2 intrastrand guanine-guanine [CP-d(GpG)] cross link was likely responsible for much of the cytotoxic actions of the compound. But current techniques prevented accurate and specific adduct quantification using pharmacologically relevant concentrations of cisplatin. Therefore, the development of a highly sensitive and specific method to measure the CPd( GpG) cross link was begun. Using this technique, this dissertation aimed to study the role of CP-d(GpG) in acquired resistance and different genetic profiles. The developed mass spectrometry method is able to measure 3.7 adducts per 108 nucleotides using 25 μg of DNA per injection. Preliminary results indicated that the method was sensitive enough to quantify adducts in ovarian carcinoma cells using as little as 12.5 μM cisplatin. It was also able to quantify adducts the kidney, liver and colon tissues of mice that had been iii given 7 mg/kg cisplatin by i.p. injection. Our hypothesis was that the density of CP-d(GpG) cross links would serve as a useful biomarker for efficacy and/or toxicity of cisplatin. Research was conducted to understand CPd( GpG) formation in ovarian carcinoma cell lines as well as in 8 inbred strains of mice. Results indicate that the dose response relationship for adduct formation in our isogenic cisplatin sensitive and resistant cell lines remains linear, when using lower more pharmacologically relevant doses of cisplatin. In mice, adducts were most concentrated in the kidney. Of the 8 inbred strains tested the C57BL/6J mice were the most sensitive and FVB/NJ least sensitive to cisplatin treatment. Toxicity, as determined by histopathology, did not correlate with CP-d(GpG) molecular dosimetry. However, this lack of correlation may be due to the design of the mouse study, as such many suggestions for future animal studies are given. Based on reported concentrations of platinum DNA adducts clinical samples, the sensitivity and specificity of our method could provide additional insight as to the role of CPd( GpG) adduct formation in cancer patients being treated with cisplatin.

Platinum chemotherapeutic agents have been widely used in the treatment of cancer. Cisplatin was the first of the platinum based chemotherapeutic agents and therefore has been extensively studied as an anti-tumor agent since the late 1960s. Because this agent forms several DNA adducts, a highly sensitive and specific quantitative assay is needed to correlate the molecular dose of individual adducts with the effects of treatment. An ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) assay for quantification of 1,2 guanine-guanine intrastrand cisplatin adducts [CP-d(GpG)], using 15N10 CP-d(GpG) as an internal standard, was developed. The internal standard was characterized by MS/MS and its concentration was validated by ICP-MS. Samples containing CP-d(GpG) in DNA were purified by enzyme hydrolysis , centrifugal filtration and HPLC with fraction collection prior to quantification by UPLC-MS/MS in the selective reaction monitoring (SRM) mode (m/z 412.5→248.1 for CP-d(GpG); m/z 417.5→253.1 for [15N10] CP-d(GpG)). Recovery of standards was >90% and quantification was unaffected by increasing concentrations of calf thymus DNA. This method utilizes 25 μg of DNA per injection. The limit of quantification was 3 fmol or 3.7 adducts per 108 nucleotides, which approaches the sensitivity of the 32P postlabeling method for this adduct. These data suggested that this method is suitable for in vitro and in vivo assessment of CP-d(GpG) adducts formed by cisplatin and carboplatin. Subsequently the method was applied to studies using ovarian carcinoma cell lines and C57/BL6 mice to illustrate that this method is capable of quantifying CP-d(GpG) adducts using biologically relevant systems and doses. The development of biomarkers to determine tissue-specific molecular dosimetry during treatment will lead to a more complete understanding of both therapeutic and adverse effects of cisplatin and carboplatin. This will support the refinement of therapeutic regimes and appropriate individualized treatment protocols.