Modified immunoenriched 32P-HPLC assay for the detection of O4-ethylthymidine in human biomonitoring studies

• Verfasst von: Godschalk, Roger [VerfasserIn]
• Verfasst von: Nair, Jagadeesan [VerfasserIn]
• Verfasst von: Kliem, Hans-Christian [VerfasserIn]
• Verfasst von: Wießler, Manfred [VerfasserIn]
• Verfasst von: Bouvier, Guy [VerfasserIn]
• Verfasst von: Bartsch, Helmut [VerfasserIn]
• Titel: Modified immunoenriched 32P-HPLC assay for the detection of O4-ethylthymidine in human biomonitoring studies
• Verf.angabe: Roger Godschalk, Jagadeesan Nair, Hans-Christian Kliem, Manfred Wiessler, Guy Bouvier, and Helmut Bartsch
• E-Jahr: 2002
• Jahr: February 22, 2002
• Umfang: 5 S.
• Fussnoten: Im Titel ist "32" bei P-HPLC hochgestellt, "4" bei O hochgestellt ; Gesehen am 12.01.2021
• Titel Quelle: Enthalten in: Chemical research in toxicology
• Ort Quelle: New York, NY : ACS Publ., 1988
• Jahr Quelle: 2002
• Band/Heft Quelle: 15(2002), 3, Seite 433-437
• ISSN Quelle: 1520-5010
• DOI: doi:10.1021/tx015582s
• Datenträger: Online-Ressource
• Sprache: eng
• K10plus-PPN: 1744424055

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 32P-postlabeling assay for O4-ethylthymidine (O4-etT) was developed. O4-etT-3‘-monophosphate (O4-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 O4-etT-3‘P using specific monoclonal antibodies. The immunoconjugate was washed by filtration, and O4-etT-3‘P was recovered by ethanol treatment. The enriched O4-etT-3‘P was labeled with [γ-32P]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, O4-etT levels were determined in calf thymus DNA treated with N-ethyl-N-nitrosourea, and a dose-dependent formation of O4-etT was observed. Furthermore, O4-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. O4-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 O4-etT in surrogate cells from cigarette smoke exposed humans.