DETECTION OF MODIFIED DNA NUCLEOTIDES BY POSTLABELING PROCEDURES

During the last 15 years the number of studies using postlabeling tech niques to detect molecular alterations in DNA exposed to genotoxic age nts has been continuously growing Detectable molecules no longer inclu de only bulky adducts arising from environmental exposures to polycycl ic aromatic hydrocarbons, heterocyclic aromatic amines, cigarette smok e, etc. Postlabeling procedures are also able to reveal small DNA addu cts and small nucleotide alterations induced by genotoxic agents such as alkylating compounds and aflatoxins. DNA alterations induced by oxi dizing molecules both of exogenous and endogenous source can also be r evealed. Postlabeling methods have been successfully used to detect DN A damage induced by ionizing and exciting radiations. Variants of the basic procedure allow the detection of endogenous DNA modifications as sociated with aging, called I-compounds. Postlabeling methods are able to detect such a great variety of molecular alterations by using a ne gligible amount of DNA (15 mu g) with an extraordinary high sensitivit y (up to 1/10(10) modified nucleotide/normal nucleotides). Different m odifications of the basic procedure are applied depending on the speci fic nucleotidic modification under analysis. The present article descr ibes the main methodological variants of postlabeling techniques, with particular attention paid to their methodological aspects, applicatio ns, and capabilities. Each step of the postlabeling procedure (i.e., D NA depolymerization, adduct enrichment, labeling, and identification) is described and the mast useful variants currently available are repo rted. DNA depolymerization may be performed by using at least 5 differ ent nucleases to obtain. biphosphate mononucleotide, monophosphate mon onucleotide, dinucleotides or trinucleotides. Modified nucleotides can be selected from among normal nucleotides by enrichment procedures in cluding more than 6 different chemical or enzymatic methods. Adducts a re labeled by using radioactive carriers such as AT(32)P and other rad ioisotopes (P-33, S-35) or fluorochromes (dansyl chloride). Labeled ad ducts are separated by thin-layer or column chromatography using a gre at variety of chromatographic media. Finally, modified nucleotides are revealed and quantified by various techniques, including standard, la ser scan, and electronic autoradiography Thus, the postlabeling proced ure no longer can be considered a single toxicologic method. It is a c lass of analytical tools able to detect a wide variety of nucleotidic modifications induced by genotoxic agents.