One-milliliter wet-digestion for inductively coupled plasma mass spectrometry (ICP-MS): determination of platinum-DNA adducts in cells treated with platinum(II) complexes.
Platinum (Pt)-DNA adducts formed by the anti-tumor agent cisplatin are recognized by the DNA mismatch repair (MMR) system. To investigate the involvement of MMR proteins including hMLH1 in the removal of these adducts, we developed a mL-scale wet-digestion method for inductively coupled plasma mass spectrometry (ICP-MS). The detection limit was 0.01 ng mL(-1) Pt, which corresponded to 2 pg Pt/microg DNA when 10 microg of DNA was used. The mean relative errors were 5.4% or better for a dynamic range of 0.01-10 ng mL(-1) Pt. DNA (approximately 500 microg) had no matrix effect. To improve the accuracy, DNA preparations were treated with ribonuclease and the apparent reduction in the concentration of Pt was corrected using cellular DNA levels, which were determined with Hoechst 33258. No significant differences were observed, in terms of the formation of Pt-DNA adducts or their removal over 6 h, between hMLH1-deficient HCT116 cells, a human colorectal cancer cell line, and hMLH1-complemented HCT116+ch3 cells (n=5; P>0.05), indicating that the hMLH1-dependent DNA repair systems contribute to neither the formation nor the removal of the adducts at detectable levels. In addition, approximately 19% of the adducts were removed within 6 h in both cell lines. A time course analysis (~24 h) suggested that the removal of cisplatin-generated Pt-DNA adducts follows first-order kinetics (t(1/2)=32 h). The amount of Pt-DNA adduct formed by oxaliplatin in 1 h was 56% (ratio of means) of that generated by an equimolar concentration of cisplatin in HCT116. The proposed procedure could be useful for determining Pt-DNA adducts formed by Pt(II) complexes.
Yamada
Kanae
K
Department of Molecular Biology, Faculty of Medicine, Toho University, 5-21-16 Ohmori-Nishi, Ohta-ku, Tokyo, 143-8540, Japan.
Kato
Naoyuki
N
Takagi
Akimitsu
A
Koi
Minoru
M
Hemmi
Hiromichi
H
D048868:Adaptor Proteins, Signal Transducing; D000970:Antineoplastic Agents / Q000378:metabolism* / Q000494:pharmacology; D002352:Carrier Proteins / Q000378:metabolism*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D002945:Cisplatin / Q000032:analysis*; D018736:DNA Adducts / Q000032:analysis*; D004260:DNA Repair; D004273:DNA, Neoplasm / Q000378:metabolism*; D006801:Humans; D013058:Mass Spectrometry; D000070957:MutL Protein Homolog 1; D009687:Nuclear Proteins / Q000378:metabolism*; D009944:Organoplatinum Compounds / Q000378:metabolism* / Q000494:pharmacology; D000077150:Oxaliplatin; D010984:Platinum / Q000032:analysis*; D012680:Sensitivity and Specificity; D013054:Spectrophotometry, Atomic
D048868:Adaptor Proteins, Signal Transducing; D000970:Antineoplastic Agents; D002352:Carrier Proteins; D018736:DNA Adducts; D004273:DNA, Neoplasm; C501486:MLH1 protein, human; D009687:Nuclear Proteins; D009944:Organoplatinum Compounds; C057270:cisplatin-DNA adduct; D000077150:Oxaliplatin; D010984:Platinum; D000070957:MutL Protein Homolog 1; D002945:Cisplatin