title: A comprehensive quantitative analysis of methylated and ethylated DNA using high pressure liquid chromatography.
abstract: Methods were developed for the efficient routine degradation and fractionation of ethylated and methylated DNA. Alkylated DNA was hydrolyzed by a neutral thermal method to yield 3- and 7- alkylpurines and O2-alkylcytosines. The partially apurinic DNA was separated from the bases by precipitation in 0.1 N HCl. Portions of the DNA precipitate were further hydrolyzed either by 0.1 N HCl to yield purine bases, or by enzymes to yield nucleosides and phosphotriesters. The chemical and enzymic digests were fractionated by a combination of high pressure liquid chromatography systems to yield quantitative estimates of the following products from methylated or ethylated DNA: 1-, 3-, and 7-alkyladenines, O2-alkylcytosines, 3-, O6-, and 7- alkylguanines and O2-, 3-, and O4-alkylthymines. N6-Alkyladenines, 1-alkylguanines and N2-alkylguanines were not detected and the 3- alkylcytosines were detected but not quantified. Phosphotriesters were estimated from the amounts of recovered alkyl phosphotriesters of thymidylyl (3'-5') thymidine. Using these methods, it was possible to account for 98, 81, 98, and 92% of the DNA bound alkyl groups obtained from DNA reacted with [14C]methyl methanesulfonate, [3H]ethyl methanesulfonate, N-[3H]-methyl-N-nitrosourea, and N-[14C]ethyl-N-nitrosourea, respectively. The methods described provide reproducible and quantitative methods of analysis for all the known methylated or ethylated products in a single DNA sample.
authors.affiliation: Department of Health and Human Services, Food and Drug Administration, National Center for Toxicological Research, Jefferson, AR 72079, USA.
authors.forename: D T
authors.initials: DT
authors.lastname: Beranek
authors.forename: C C
authors.initials: CC
authors.lastname: Weis
authors.forename: D H
authors.initials: DH
authors.lastname: Swenson
mesh_terms: D000477:Alkylating Agents / Q000494:pharmacology; D000478:Alkylation; D000818:Animals; D002273:Carcinogens / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002852:Chromatography, Ion Exchange; D004247:DNA / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D019175:DNA Methylation; D006868:Hydrolysis; D008297:Male; D010755:Organophosphates / Q000032:analysis / Q000737:chemistry; D015203:Reproducibility of Results; D012474:Salmon; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism; D013094:Spermatozoa / Q000737:chemistry
chemical_list: D000477:Alkylating Agents; D002273:Carcinogens; D010755:Organophosphates; D004247:DNA; D015719:Single-Strand Specific DNA and RNA Endonucleases