Cisplatin DNA Adduct Detection and Depurination Measured by³²P DNA Radiolabeling and Two-Dimensional Thin-Layer Chromatography: A Time and Concentration Study

Abstract

Platinum-based chemotherapies cause the formation of DNA adducts and have profound effects on DNA. This study measured cis-diamminedichloroplatinum II (cisplatin) DNA adducts by 32P-radiolabeling DNA, enzymatically digesting radiolabeled DNA, separating the formed adducts on two-dimensional thin-layer chromatography, and quantitating the adducts with autoradiography and densitometry. HeLa DNA was incubated with cisplatin at varying concentrations (6.25-325 nM) and times (0 min to 72 hr). Cisplatin rapidly depurinated dGMP and dAMP (90%, 15-min incubation with 325 nM cisplatin). Partial depurination of dGMP (15%) and dAMP (25%) occurred with lower cisplatin concentrations at equal incubation times. A minimum of four new adducts, with relatively rapid migratory patterns, were detected at high cisplatin concentrations with short incubation times. These results indicate that the depurination of DNA correlates with DNA adduct formation and that the quantification of these adducts may be applicable to monitoring tumor and host cell response to cisplatin chemotherapy.

Nucleic Acids and Their Derivatives

“…Exposure times enhance the ability to label unknowns at high counts in as little as 2 h, but typically run 24-72 h. Present densitometric techniques can range from a few minutes for one-dimensional analyses to 2 h for complete analyses of a 20 X 20 cm autoradiogram. Comparisons of techniques to direct scintillation counting approach r values of 0.99 and similarly correlate with the best quantifying techniques (7)(8)(9)12,50,55).…”

“…It is extremely easy to compare prior TLC plates, generate tables for comparison, and rapidly apply statistical or analytical methods by computer. This has yielded quantifiable relationships in nucleic acid and DNA chemistry that were difficult to examine previously (7)(8)(9)12,50,55).…”

“…The better ability to subtract controls, carry out statistical analyses, and enhance minute adducts magnifies adduct and analog detection of nucleic acids. Some studies have presented data on phosphoroimager use and TLC including "high power" focusing carried out by computer interaction (7)(8)(9)12,50,55).…”

“…Thin-layer chromatography has been used in the study of metabolic diseases; illicit drug use; toxicology, including mycotoxins; environmental injury; and, particularly, analytical drug characterization and quantification for both diagnostic and regulatory purposes (7)(8)(9)12,50,55,62,63). It has extensive applications for the pharmaceutical industry in all areas of drug development: synthesis, separation, drug screening, batch quality testing, and government monitoring and reg- ulations.…”

“…Mass spectrometry (MS) is used for nucleic acids, but typically after zonal elution to avoid interfering solutes (8,61). Ion-coupled MS/TLC must take into account the sorbent, solvent, and analyte, which should not exceed 0.25%, (w/w) based on sample and sorbent.…”