International Union of Pure and Applied Chemistry Analytical Chemistry Division Commission on Radiochemistry and Nuclear Techniques* Isotopic and Nuclear Analytical Techniques in Biological Systems a Critical Survey

Covalent binding of chemicals or their metabolites to DNA is a critical event in the initiation of chemical carcinogenesis. Measurement of covalent DNA adducts in model and human systems has become an important tool in mutation and cancer research. The recently developed , highly sensitive 32P-postlabeling assay of DNA adducts and its applications are critically reviewed. Basic features of assay 32P-postlabeling analysis is a recently developed, highly sensitive method for the detection and measurement of covalent DNA adducts. Since the assay is based on the enzymatic incorporation of 32P into DNA nucleotides, it does not require the carcinogens to be radiolabeled. Therefore, the method is suitable for DNA of humans exposed to environmental or occupational genotoxicants. The basic method (ref. 1) entails digestion of DNA with a mixture of micrococcal nuclease and spleen phosphodiesterase to 3'mononucleotides , conversion of the di estion products to 5'-32P-labeled 3',5'-bisphosphate derivatives with [y-'P]ATP as the donor of label and T4 polynucleotide kinase as the catalyst, separation of the labeled nucleotides by polyethyleneimine (PE1)-cellulose TLC, autoradiography to detect adducts and normal nucleotides, and quantitation by scintillation counting. A key feature of the assay is the class separation of aromatic or bulky/hydrophobic adducts from the usually large excess of normal DNA nucleotides; this is readily accomplished by PEI-cellulose TLC (refs. 2-6). Various versions of the assay have been developed. The adducts may be labeled in the presence of the normal DNA nucleotides, or, alternatively, to increase the sensitivity of the assay, normal nucleotides are removed before 32P-labeling of the adducts. For adduct enrichment, physical techniques such as butanol extraction (ref. 7) and HPLC (refs. 8, 9) or enzymatic techniques such as nuclease P1 post-digestion (ref. 10) have been described. In the nucleoside monophosphate postlabeling assay (ref. ll), adducts are purified as part of the initial DNA hydrolysis step and obtained in the form of dinucleotides which are subsequently converted to 32P-labeled 1284 COMMISSION ON RADIOCHEMISTRY AND NUCLEAR TECHNIQUES 5r-monophosphates. The enrichment procedures afford the greatest sensitivity of adduct detection (1 adduct in lo9 10" DNA nucleotides) and usually require 5-10 pg DNA per individual assay. Recoveries of certain adducts by the enrichment procedures depend on adduct structure (refs. 10-12).