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摘要
This paper describes methodologies, based on sector field inductively-coupled plasma mass spectrometry (SF-ICP-MS), and their application in the holistic study of the fate of Pt in human cell populations following treatment with cis- or oxaliplatin and combination treatments. Pt-DNA adduct formation data at several time points has been determined in the leukocytes from patients undergoing Pt-based chemotherapy demonstrating significant inter-patient variability and excellent reproducibility of the assay. The sensitivity of the technique enabled quantitation of as little as 0.2 Pt adducts per 10(6) nucleotides using 10 mu g of patient DNA. Further, the first ever reported in vivo sub-cellular Pt fractionation data on a patient sample is presented indicating the feasibility of applying the methods presented here in a clinical environment. For in vitro studies, three cell models were used: A549 human lung adenocarcinoma epithelial cells were exposed to 50 mu M cisplatin for 1 h; HCA7 human colorectal cancer cells were treated with either FOLFOX (200 mu M 5-fluorouracil, 200 mu M folinic acid and 50 mu M oxaliplatin) or 50 mu M oxaliplatin; and HT29 human colorectal cancer cells were treated with 50 mu M oxaliplatin in combination with 20 mu M methaneseleninic acid, CH(3)SeO(2)H (MSA). The cells were harvested and either the DNA extracted and/or a commercially available kit used to fractionate the treated cells into four sub-cellular compartments. Each of the sub-cellular fractions and extracted DNA were digested separately, evaporated to dryness and reconstituted in 2% nitric acid for analysis by SF-ICP-MS. The sub-cellular Pt distribution for cisplatin treated A549 cells was shown to be as follows: similar to 70% localized in the cytosol, similar to 17% in the membrane and membrane localized fraction, similar to 9% in the nuclear fraction and similar to 4% in the cytoskeletal fraction. Both FOLFOX and oxaliplatin treated HCA7 cells showed comparable sub-cellular Pt distributions, and Pt-DNA adduct formation was similar for the oxaliplatin and FOLFOX treatments with adduct yields of 5.6 and 5.5 adducts per 10(6) nucleotides respectively. It was found that the combination of oxaliplatin with 20 mu M MSA did not change the distribution of Pt or significantly alter its accumulation in the cytosol of the HT29 cells. Mass balance experiments showed a >99% recovery of the total Pt in the sub-cellular fractions. These experiments are the first to provide such a detailed quantitation of Pt-drug partitioning and they show that the Pt broadly follows the total protein content of the individual compartments with the majority being scavenged in the cytosol compartment.
- 出版日期2011-10-1
