POSTLABELING DETECTION OF DNA-ADDUCTS OF ANTITUMOR ALKYLATING-AGENTS

The sensitivity for DNA adduct formation by antitumor alkylating agent s (mechlorethamine, cisplatin and adozelesin) of the postlabeling tech nique and thin-layer chromatography was studied. Three DNAs were used: a double-stranded 20-bp oligonucleotide of defined sequence, calf thy mus DNA and murine leukemia L1210 cellular DNA. With high concentratio ns of mechlorethamine, there was a marked decrease in normal dGp, a le sser decrease in dAp and dCp and no change in dTp. Using 2D mapping PE I-cellulose thin-layer chromatography analyses, it was found that six mechlorethamine: DNA adducts were produced after a short exposure to m echlorethamine. After an extended time at relatively high drug concent rations there was an alteration in the mechlorethamine: DNA adduct pat tern that may reflect the conversion of monoadducts to crosslinked add ucts. Similar observations were made with cisplatin and adozelesin. Wh en murine leukemia L1210 cells were treated with 50 mu M mechlorethami ne or 50 mu M cisplatin for 1 h, six or more mechlorethamine: DNA addu cts and fire cisplatin: DNA adducts were detected. After allowing 6 h. for repair of potentially lethal damage, several adducts were no long er detectable and others appeared with diminished intensity. Nuclease P-1 dephosphorylates normal nucleotides at relatively low enzyme conce ntrations with variation depending upon the nucleotide. In general, co nsiderably lower concentrations of nuclease P-1 were required to depho sphorylate the normal nucleotides than to dephosphorylate the antitumo r alkylating agent: nucleotide adducts, thus allowing increased sensit ivity of the postlabeling assay. The sensitivity of detection of antit umor alkylating agent: DNA adducts in DNA from treated L1210 cells app roached one adduct per 10(7)-10(8) nucleotides. These results suggest that the postlabeling technique may be sufficiently sensitive and spec ific for the study of the clinically effective levels of antitumor alk ylating agents.