Determination of Cisplatin 1,2-Intrastrand Guanine-Guanine DNA Adducts in Human Leukocytes by High-Performance Liquid Chromatography Coupled to Inductively Coupled Plasma Mass Spectrometry
Determination of Cisplatin 1,2-Intrastrand Guanine-Guanine DNA Adducts in Human Leukocytes by High-Performance Liquid Chromatography Coupled to Inductively Coupled Plasma Mass Spectrometry
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DOI：
10.1021/tx100023c
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发表时间：
2010-08-01
影响因子：
4.1
通讯作者：
Farmer, Peter B.
中科院分区：
文献类型：
--
作者：
Harrington, Chris F.;Le Pla, Rachel C.;Farmer, Peter B.
含铂类药物在各种临床环境中广泛用于治疗癌症。它们的作用模式涉及DNA加合物的形成，从而促进癌细胞的凋亡。顺铂结合嘌呤DNA碱基的N7位置，在相邻的两个鸟嘌呤[顺式- pt (NH(3))(2)d(pGpG), 1,2- gg]或相邻的腺嘌呤和鸟嘌呤[顺式- pt (NH(3))(2)d(pApG), 1,2- ag]之间形成链内交联。每一种不同类型的DNA加合物的细胞毒性作用以及肿瘤细胞和血液中加合物水平之间的关系尚不清楚。通过使用这些含pt的加合物作为生物标志物，患者对化疗反应的信息将与药物的作用方式直接相关。这种类型的分析需要最敏感和最具体的方法，以促进检测限，足以测量从患者可获得的有限样品数量中的DNA加合物。本研究通过将一种高度特异性的酶加合物分离方法与一种基于高效液相色谱耦合到电感耦合等离子体质谱的灵敏检测系统相结合，来测量DNA中形成的I，2-GG顺铂加合物，从而实现了这一目标。利用小牛胸腺DNA和两种不同的腺癌细胞系开发并验证了该方法。1,2- gg顺铂加合物的检出限和定量限分别为0.21和0.67 fmol / μ g DNA。这校正了1,2- gg加合物的绝对LOD为0.8 pg / Pt。顺铂敏感（H23）和耐药（A549）肿瘤细胞暴露于药物，并在24小时内测量1,2- gg加合物水平。结果显示，修复7 h后，敏感细胞的加合物浓度明显高于抗性细胞（P < 0.05）。尽管在随后的时间点（12和24 h），加合物浓度有所下降，但各细胞系的加合物水平大致相似。然后将该方案应用于分析治疗前和治疗后1小时采集的患者样本。在治疗后采集的所有患者样本中，1,2- gg顺铂加合物在113 - 1245 fg Pt / mu g DNA范围内存在。虽然在最初的预处理样品中，加合物的含量不高于LOD，但在第三个治疗周期的一些患者样品中，可以看出痕量的加合物。
Platinum-containing drugs are widely used to treat cancer in a variety of clinical settings. Their mode of action involves the formation of DNA adducts, which facilitate apoptosis in cancer cells. Cisplatin binds to the N7 position of the purine DNA bases forming intrastrand cross-links between either two adjacent guanines [cis-Pt(NH(3))(2)d(pGpG), 1,2-GG] or an adjacent adenine and guanine [cis-Pt(NH(3))(2)d(pApG), 1,2-AG)]. The cytotoxic efficacy for each of the different types of DNA adducts and the relationship between adduct levels in tumor cells and blood are not well understood. By using these Pt-containing adduct species as biomarkers, information on a patient's response to chemotherapy would be directly related to the mode of action of the drug. This type of analysis requires the most sensitive and specific methods available, to facilitate detection limits sufficient to measure the DNA adduct in the limited sample quantities available from patients. This was achieved in the current study by coupling a highly specific enzyme-based adduct isolation method with a sensitive detection system based on HPLC coupled to inductively coupled plasma mass spectrometry to measure the I,2-GG cisplatin adducts formed in DNA. The method was developed and validated using calf thymus DNA and two different adenocarcinoma cell lines. The values for the limit of detection (LOD) and the limit of quantitation determined for the 1,2-GG cisplatin adduct were 0.21 and 0.67 fmol per mu g DNA, respectively. This correcponds to an absolute LOD of 0.8 pg as Pt for the 1,2-GG adduct. Cisplatin-sensitive (H23) and -resistant (A549) tumor cells were exposed to the drug, and the 1,2-GG adduct levels were measured over a 24 h tithe period. The results showed a statistically significant (P < 0.05) higher concentration in the sensitive cells as compared to the resistant cells after repair for 7 h. Although the adduct concentration present fell at subsequent time points (12 and 24 h), the levels in each cell line were broadly similar. The protocol was then applied to the analysis of patient samples taken before and then 1 h after treatment. The 1,2-GG cisplatin adduct was present in the range from 113 to 1245 fg Pt per mu g DNA in all of the patient samples taken after treatment. Although the adduct was not present at levels greater than the LOD in the initial pretreatment samples, trace amounts were discernible in some patient samples on their third treatment cycle.