Detection of modified DNA nucleotides by postlabeling procedures

During the last 15 years the number of studies using postlabeling techniques to detect molecular alterations in DNA exposed to genotoxic agents has been continuously growing Detectable molecules no longer include only bulky adducts arising from environmental exposures to polycyclic aromatic hydrocarbons, heterocyclic aromatic amines, cigarette smoke, etc. Postlabeling procedures are also able to reveal small DNA adducts and small nucleotide alterations induced by genotoxic agents such as alkylating compounds and aflatoxins. DNA alterations induced by oxidizing molecules both of exogenous and endogenous source can also be revealed. Postlabeling methods have been successfully used to detect DNA damage induced by ionizing and exciting radiations. Variants of the basic procedure allow the detection of endogenous DNA modifications associated with aging, called I-compounds. Postlabeling methods are able to detect such a great variety of molecular alterations by using a negligible amount of DNA (15 mu g) with an extraordinary high sensitivity (up to 1/10(10) modified nucleotide/normal nucleotides). Different modifications of the basic procedure are applied depending on the specific nucleotidic modification under analysis. The present article describes the main methodological variants of postlabeling techniques, with particular attention paid to their methodological aspects, applications, and capabilities. Each step of the postlabeling procedure (i.e., DNA depolymerization, adduct enrichment, labeling, and identification) is described and the mast useful variants currently available are reported. DNA depolymerization may be performed by using at least 5 different nucleases to obtain. biphosphate mononucleotide, monophosphate mononucleotide, dinucleotides or trinucleotides. Modified nucleotides can be selected from among normal nucleotides by enrichment procedures including more than 6 different chemical or enzymatic methods. Adducts are labeled by using radioactive carriers such as AT(32)P and other radioisotopes (P-33, S-35) or fluorochromes (dansyl chloride). Labeled adducts are separated by thin-layer or column chromatography using a great variety of chromatographic media. Finally, modified nucleotides are revealed and quantified by various techniques, including standard, laser scan, and electronic autoradiography Thus, the postlabeling procedure no longer can be considered a single toxicologic method. It is a class of analytical tools able to detect a wide variety of nucleotidic modifications induced by genotoxic agents.