Detection of DNA alkylphosphotriesters by P-32 postlabeling: Evidence for the nonrandom manifestation of phosphotriester lesions in vivo

Many genotoxic carcinogens react with the sugar-phosphate backbone in DNA t o form phosphotriester (PTE) adducts, These lesions are relatively abundant and persistent for some alkylating carcinogens and may therefore serve as useful biomarkers with which to assess genotoxic exposure and potential mut agenic risk. In the present study, we have developed a P-32 postlabeling me thod that permits analysis of total methyl and/or ethyl PTE in DNA at the f emtomole level. The technique is based on the inability of all known nucleo lytic enzymes to cleave the internucleotide PTE bond. Consequently, complet e digestion of alkylated DNA with these nucleases in the presence of an alk aline phosphatase yields PTE-dinucleoside phosphates. These species are the n converted to the corresponding dinucleoside phosphates (dNpdNs) by treatm ent with alkali to permit subsequent P-32 labeling. The resulting labeled d inucleotides ((32)pdNpdN) are then analyzed by PAGE. Validation of this met hod has been carried out using a polydeoxythymidylic acid oligonucleotide c ontaining a site-specific methyl PTE. The method has been applied to the in vitro analysis of calf thymus (CT) DNA treated with dimethylsulfate (DMS) or diethylsuifate (DES) and to the analysis of liver DNA from mice treated in vivo with nitrosodiethylamine. In each case, autoradiograms of the polya crylamide gels showed the anticipated five bands representing the sixteen l abeled dinucleotides, with proportional increases observed as the concentra tions of DMS or DES used in the in vitro treatment of CT DNA were increased . The identity and frequency of the nucleosides located 5' to the PTE lesio ns were obtained by nuclease P1 digestion of the gel-isolated (32)pdNpdN sp ecies and by analysis of the released labeled mononucleotides, (32)pdN, by high-performance liquid chromatography with radioactivity detection, Result s obtained from CT DNA treated with DMS or DES showed that the frequency of the four detected nucleotides reflected the normal nucleoside content of C T DNA, indicating the random formation of methyl and ethyl PTE adducts in t he in vitro modified DNA, However, studies using liver DNA from three strai ns of mice treated in vivo with nitrosodiethylamine indicated that the freq uency of the thymidine and the 2'-deoxyguanosine 5' to the ethyl PTE was si gnificantly different from the corresponding normal nucleoside content. The se results are indicative of (a) the nonrandom formation of ethyl PTE in vi vo and/or (b) base sequence-specific ethyl PTE repair.