16174-59-7Relevant articles and documents
32P-Postlabeling of N-(deoxyguanosin-8-yl)arylamine adducts: a comparative study of labeling efficiencies.
Mourato,Beland,Marques
, p. 661 - 669 (1999)
32P-Postlabeling is an extremely powerful technique for the detection of DNA adducts. Typically, the quantitation of DNA adducts by (32)P-postlabeling is achieved by relative adduct labeling, via comparison of the radioactivity incorporated into the adducts to that associated with the normal nucleotides. This approach is based on a number of assumptions, the foremost being that normal and adducted nucleotide 3'-phosphates are converted to 3', 5'-bisphosphates with similar efficiencies. To evaluate labeling efficiencies for specific DNA adducts, we conducted a comparative study of the kinetics of phosphorylation by T(4) polynucleotide kinase using 2'-deoxyguanosine 3'-phosphate (dG3'p) and a series of N-(deoxyguanosin-8-yl)arylamine 3'-phosphate adduct standards (dG3'p-C8-Ar, Ar being 4-aminobiphenyl, 3- and 4-methylaniline, and 2,4- and 3,4-dimethylaniline). Phosphorylation of dG3'p and the dG3'p-C8-Ar adducts followed Michaelis-Menten kinetics. The apparent turnover numbers were 40-240-fold lower when labeling dG3'p-C8-Ar adducts compared to that when labeling dG3'p. The apparent specificity constant calculated for dG3'p-C8-4-aminobiphenyl (1.4 microM(-)(1) min(-)(1)) was approximately 4-fold lower than that (5. 4 microM(-)(1) min(-)(1)) found for dG3'p. Apparent specificity constants for the monoarylamine adducts were even lower (0.043-0.23 microM(-)(1) min(-)(1)) and decreased in the following order: 4-methylaniline > 3,4-dimethylaniline > 3-methylaniline > 2, 4-dimethylaniline. Similar experiments conducted with dG3'p-C8-Ar standards for 2-methylaniline and 2,3-dimethylaniline produced very poor and irreproducible labeling. These results indicate that (32)P-postlabeling of dG3'p-C8-Ar adducts is less efficient than that of dG3'p and suggest that normal nucleotides will be labeled preferentially to the arylamine adducts under kinetically controlled conditions. The data also indicate a further decrease in labeling efficiency upon substitution ortho to the amino group (e.g., 2, 4-dimethylaniline). In addition, the ATP concentrations required for optimal labeling were found to be substantially higher than those used in typical (32)P-postlabeling assays. Since the high specific activity of carrier-free [gamma-(32)P]ATP precludes increasing the ATP concentration to a significant extent, these data emphasize the need for using highly efficient adduct enrichment procedures when conducting (32)P-postlabeling analyses of DNA adducts.
Bennett et al.
, p. 4623,4624,4626 (1976)
Carcinogen-DNA adducts in human breast epithelial cells
Gorlewska-Roberts,Green,Fares,Ambrosone,Kadlubar
, p. 184 - 192 (2007/10/03)
Diet and environmental exposures are often regarded as significant etiologic factors in human breast cancer. Chemicals that may be involved in these exposures include heterocyclic amines, aromatic amines, and polycyclic aromatic hydrocarbons, which also serve as strong mammary carcinogens in different animal models. In this study, we chose to quantify the major DNA adducts derived from one member of each of these classes of carcinogens, that is, 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP), 4-aminobiphenyl (ABP), and benzo[a]pyrene (B[a]P), respectively, in DNA isolated from exfoliated ductal epithelial cells in human breast milk. Milk was collected from healthy, nonsmoking mothers. The isolated DNA was digested to 3′ nucleotides and subjected to 32P-postlabeling. Adduct enrichment was achieved using Oasis Sep-Paks and the analyses were conducted by HPLC using radiometric detection. Critical to the analyses were the syntheses of bis(phosphate) standards for the C8-dG adducts of PhIP and ABP, and the N2-dG adduct of B[a]P, which were added to each reaction as UV markers. Of the 64 samples analyzed, adducts were found in 31 samples. Thirty samples contained detectable levels of PhIP adducts, with a mean value of 4.7 adducts/107 nucleotides; 18 were positive for ABP adducts with a mean value of 4.7 adducts/ 107 nucleotides; and 13 were bund to contain B[a]P adducts with a mean level of 1.9 adducts/ 107 nucleotides. These data indicate that women are exposed to several classes of dietary and environmental carcinogens and that these carcinogens react with DNA in breast ductal epithelial cells, the cells from which most breast cancers arise.
