High-resolution TLC mapping and characterization of 32P-postlabeled monophosphate 7,12-dimethylbenz[a]anthracene-DNA adducts.
Previous work has shown that 7,12-dimethylbenz[a]anthracene (DMBA)-DNA adducts can be converted by 32P-postlabeling to different types of radiolabeled derivatives (nucleoside 3',5'-bisphosphates, nucleoside 5'-monophosphates and dinucleotides), and that the 32P-labeled 3',5'-bisphosphate derivatives can be further characterized by cross-referencing with 3H-labeled nucleoside DMBA adducts, for which structural information is available. This work has now been extended by TLC comparisons of 5'-monophosphate and 3',5'-bisphosphate DMBA adducts. To this end, DMBA-modified DNA was enzymatically hydrolyzed to 3'-monophosphates (Xp + Np) or to dinucleotides (XpN), and these digestion products were 32P-postlabeled by published procedures to yield 3',5'-bisphosphate (*pXp) or 5'-monophosphate (*pX) adducts. Individual *pXp and *pX fractions were isolated from polyethyleneimine (PEI)-cellulose TLC maps and chromatographically compared after enzymatic 3'-dephosphorylations of the 3',5'-bisphosphate (*pXp) derivatives (*pXp----*pX + Pi). Four reactions were standardized and employed for this purpose: (i) 3'-dephosphorylation by extensive digestion with nuclease P1; (ii) 3'-dephosphorylation catalyzed by polynucleotide kinase; (iii) partial dephosphorylation by bacterial alkaline phosphatase; and (iv) partial dephosphorylation by prostatic acid phosphatase. Individual DMBA adducts displayed marked differences with regard to their susceptibility to enzymatic dephosphorylation. The three major and most minor postlabeled 5'-monophosphate DMBA adducts were cross-referenced this way with 3',5'-bisphosphate and nucleoside adducts, so that specific dihydrodiol epoxide-nucleoside 5'-monophosphate adducts can now be identified and measured by 32P-postlabeling.
Yang
P F
PF
Department of Pharmacology, Baylor College of Medicine, Houston, TX 77030.
Randerath
K
K
D015127:9,10-Dimethyl-1,2-benzanthracene / Q000302:isolation & purification / Q000378:metabolism / Q000031:analogs & derivatives; D000818:Animals; D002855:Chromatography, Thin Layer / Q000379:methods; D004247:DNA / Q000302:isolation & purification* / Q000378:metabolism; D018736:DNA Adducts; D051379:Mice; D008813:Mice, Inbred ICR; D009710:Nucleotide Mapping; D010761:Phosphorus Radioisotopes; D011865:Radioisotope Dilution Technique; D012867:Skin / Q000378:metabolism; D012997:Solvents
C059444:7,12-dimethylbenz(a)anthracene-DNA adduct; D018736:DNA Adducts; D010761:Phosphorus Radioisotopes; D012997:Solvents; D015127:9,10-Dimethyl-1,2-benzanthracene; D004247:DNA