title: Modified immunoenriched (32)P-HPLC assay for the detection of O(4)-ethylthymidine in human biomonitoring studies.
abstract: Increased excretion of ethylated DNA bases has been reported in the urine of cigarette smokers. To study DNA ethylation in the target organs of smokers, an immunoenriched (32)P-postlabeling assay for O(4)-ethylthymidine (O(4)-etT) was developed. O(4)-etT-3'-monophosphate (O(4)-etT-3'P) was synthesized, purified, and characterized by LC-MS, ESI-MS, and NMR. DNA was enzymatically digested to 2'-deoxynucleoside-3'-monophosphate followed by immunoprecipitation of O(4)-etT-3'P using specific monoclonal antibodies. The immunoconjugate was washed by filtration, and O(4)-etT-3'P was recovered by ethanol treatment. The enriched O(4)-etT-3'P was labeled with [gamma-(32)P]ATP in the presence of T4-polynucleotide kinase at pH 6.8 to yield its 5'-labeled monophosphate and was subsequently resolved on RP-HPLC and detected with online detection of radioactivity. Adduct recovery was >80%, and the detection limit was approximately 500 amol. To further validate the method, O(4)-etT levels were determined in calf thymus DNA treated with N-ethyl-N-nitrosourea, and a dose-dependent formation of O(4)-etT was observed. Furthermore, O(4)-etT was found to be present in the cells obtained from the lower respiratory tract by sputum induction of two out of four smokers but not in three nonsmokers. O(4)-etT is a poorly repaired promutagenic DNA lesion; thus, it could be of potential use for biomonitoring smoking-related DNA damage. Our improved assay was found to be sufficiently sensitive and specific to detect O(4)-etT in surrogate cells from cigarette smoke exposed humans.
authors.affiliation: Division of Toxicology and Cancer Risk Factors, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany.
authors.forename: Roger
authors.initials: R
authors.lastname: Godschalk
authors.forename: Jagadeesan
authors.initials: J
authors.lastname: Nair
authors.forename: Hans-Christian
authors.initials: HC
authors.lastname: Kliem
authors.forename: Manfred
authors.initials: M
authors.lastname: Wiessler
authors.forename: Guy
authors.initials: G
authors.lastname: Bouvier
authors.forename: Helmut
authors.initials: H
authors.lastname: Bartsch
mesh_terms: D002273:Carcinogens; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D018736:DNA Adducts / Q000737:chemistry* / Q000302:isolation & purification; D005038:Ethylnitrosourea; D006801:Humans; D016676:Macrophages, Alveolar / Q000737:chemistry; D010761:Phosphorus Radioisotopes; D012907:Smoking / Q000276:immunology / Q000378:metabolism*; D013183:Sputum / Q000166:cytology; D013936:Thymidine / Q000031:analogs & derivatives* / Q000032:analysis*
chemical_list: D002273:Carcinogens; D018736:DNA Adducts; D010761:Phosphorus Radioisotopes; C030119:O(4)-ethylthymidine; D005038:Ethylnitrosourea; D013936:Thymidine