Detection of Sulfur Mustard-Induced DNA Damage by a 32P-Postlabeling Method

We have applied the 32p-postlabeling technique to the detection of sulfur mustard-induced damage in white blood cell DNA. Red blood cells are lysed, and DNA is released from white cells by proteinase K treatment proteins are removed by salt precipitation and DNA is collected by ethanol precipitation. Digestion to the 3-deoxynucleotide level is performed with micrococcal nuclease and spleen phosphodiesterase, and the mixture of normal and modified nucleotides is labeled by T4 kinase using Gamma-32Patp. The resulting deoxynucleoside-3,5- diphosphates are converted to deoxynucleoside-5-phosphates with PI nuclease. Most of the radioactivity can be removed by passage through a disposable anion exchange column because the major adduct, 7-hydroxyethylthioethyl deoxyguanosine 5-phosphate HETEpdG, elutes well ahead of normal deoxynucleotides and residual Gamma-32PATP. The eluent containing HETEpdG is concentrated and separated on a C-18 column by high performance liquid chromatography one min fractions are collected and counted in a scintillation counter. The 7- hydroxyethylthioethyl guanine content in the DNA is then determined from the radioactivity associated with the HETEpdG peak. Advantages of this technique include its femtomole sensitivity and its requirement for only one microgram of DNA as well as its potential ability to detect other sulfur mustard-induced DNA adducts which may have more biological significance than 7-hydroxyethylthioethyl guanine.