Molecular dosimetry of 1,2 guanine-guanine intrastrand cross links of cisplatin by ultra performance liquid chromatography tandem mass spectrometry.

摘要

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 CP-d(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 mug 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 muM cisplatin. It was also able to quantify adducts the kidney, liver and colon tissues of mice that had been 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 CP-d(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 CP-d(GpG) adduct formation in cancer patients being treated with cisplatin.