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(trans-R, R)-1,2-diaminocyclohexaneoxalatoplatinum(II) (oxaliplatin) is a recently approved platinum analogue for use in the chemotherapy of metastatic colorectal cancer. Like many cytotoxic drugs, oxaliplatin exerts its antitumor effects by covalent modification of DNA. We report an accelerator mass spectrometry (AMS) assay to measure the kinetics of oxaliplatin-induced DNA damage and repair. We determined the apparent rate of oxaliplatin adduction to salmon sperm DNA. The oxaliplatin-DNA adduct distribution was further investigated at the nucleoside level by HPLC-AMS. Cultured platinum-sensitive testicular (833K) and platinum-resistant breast and bladder (MDA-MB-231 and T24, respectively) cancer cells were incubated with a subpharmacological concentration of oxaliplatin (0.2 microM). Both cellular and DNA radiocarbon contents in the drug-sensitive testicular cells had approximately twice the area under the curve as compared to the more platinum-resistant cell lines, implying that differential accumulation of the drug may be responsible for the sensitivity of cancer cells to platinum treatment. The lowest concentration of radiocarbon measured was approximately 1+/-0.1 amol/microg of DNA, when assaying 1 microg of DNA. This sensitivity for measuring oxaliplatin-DNA adducts is the highest reported to date. The sensitivity offered by this method may be applicable to other DNA-damaging drugs, metabolisms studies, and diagnostics development.

Cisplatin and carboplatin are platinum-based drugs that are widely used in cancer chemotherapy. The cytotoxicity of these drugs is mediated by platinum-DNA monoadducts and intra- and interstrand diadducts, which are formed following uptake of the drug into the nucleus of cells. The pharmacodynamics of carboplatin display fewer side effects than for cisplatin, albeit with less potency, which may be due to differences in rates of DNA adduct formation. We report the use of accelerator mass spectrometry (AMS), a sensitive detection method often used for radiocarbon quantitation, to measure both the kinetics of [14C]carboplatin-DNA adduct formation with genomic DNA and drug uptake and DNA binding in T24 human bladder cancer cells. Only carboplatin-DNA monoadducts contain radiocarbon in the platinated DNA, which allowed for calculation of kinetic rates and concentrations within the system. The percent of radiocarbon bound to salmon sperm DNA in the form of monoadducts was measured by AMS over 24 h. Knowledge of both the starting concentration of the parent carboplatin and the concentration of radiocarbon in the DNA at a variety of time points allowed calculation of the rates of Pt-DNA monoadduct formation and conversion to toxic cross-links. Importantly, the rate of carboplatin-DNA monoadduct formation was approximately 100-fold slower than that reported for the more potent cisplatin analogue, which may explain the lower toxicity of carboplatin. T24 human bladder cancer cells were incubated with a subpharmacological dose of [14C]carboplatin, and the rate of accumulation of radiocarbon in the cells and nuclear DNA was measured by AMS. The lowest concentration of radiocarbon measured was approximately 1 amol/10 microg of DNA. This sensitivity may allow the method to be used for clinical applications.

The DNA oxidation product 7,8-dihydro-8-oxoguanine (8-oxoG) forms several mutagenic oxidation products, including a metastable oxaluric acid (Oa) derivative. We report here that a synthetic oligonucleotide containing Oa hydrolyzes under simulated "in vivo" conditions to form a mutagenic urea (Ua) lesion. Using the Oa 2'-deoxyribonucleoside as a model, the hydrolysis rate depended strongly upon the concentrations of bicarbonate and divalent magnesium. In buffered solutions containing physiologically relevant levels of these species, the half-life of Oa nucleoside was approximately 40 h at 37 degrees C. The mutagenic properties of Ua in DNA were investigated using a M13mp7L2 bacteriophage genome containing Ua at a specific site. Transfection of the lesion-containing genome into wild-type AB1157 Escherichia coli allowed determination of the mutation frequency and DNA polymerase bypass efficiency from the resulting progeny phage. Ua was bypassed with an efficiency of 11% as compared to a guanine control and caused a 99% G-->T mutation frequency, assuming the lesion originated from G, which is at least an order of magnitude higher than the mutation frequency of 8-oxoG under the same conditions. SOS induction of bypass DNA polymerase(s) in the bacteria prior to transfection caused the mutation frequency and type to shift to 43% G-->T, 46% G-->C, and 10% G-->A mutations. We suggest that Ua is instructional, meaning that the shape of the lesion and its interactions with DNA polymerases influence which nucleotide is inserted opposite the lesion during replication and that the instructional nature of the lesion is modulated by the size of the binding pocket of the DNA polymerase. Replication past Ua, when formed by hydrolysis of the 8-oxoG oxidation product Oa, denotes a pathway that nearly quantitatively generates point mutations in vivo.