Extraction and Purification of Depurinated Benzo[a]pyrene-Adducted DNA Bases from Human Urine by Immunoaffinity Chromatography Coupled with HPLC and Analysis by LC/Quadrupole Ion-Trap MS

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• 作者：Sumitra Bhattacharya ; Damon C. Barbacci ; Meilan Shen ; Jia-nuo Liu ; and George P. Casale
• 刊名：Chemical Research in Toxicology
• 出版年：2003
• 出版时间：April 2003
• 年：2003
• 卷：16
• 期：4
• 页码：479 - 486
• 全文大小：200K
• 年卷期：v.16,no.4(April 2003)
• ISSN：1520-5010

文摘

In this paper, we describe implementation and testing of an immunoaffinity (IA) column forrapid and selective extraction of 7-(benzo[a]pyren-6-yl)adenine (BP-6-N7Ade) and 7-(benzo[a]pyren-6-yl)guanine (BP-6-N7Gua) from urine, where BP is benzo[a]pyrene. The BP radicalcation is a carcinogenic metabolite that reacts with double-stranded DNA, producing depurinated BP-adducted DNA bases excreted in urine. The expected modified nucleobases are BP-6-N7Gua, BP-6-N7Ade, and 8-(benzo[a]pyren-6-yl)guanine (BP-6-C8Gua), and they may serveas important biomarkers for DNA damage by PAHs. IA extracts of urine from a cigarette smokerand a nonsmoker contained less than 5% of contaminants present in Sep-Pak extracts and,unlike the latter, were suitable for analytical HPLC. IA extraction achieved 75-95% recoveryof BP-6-N7Gua (10 fmol/mL) and BP-6-N7Ade (1 fmol/mL) added to urine samples. Tandemmass spectrometry of IA/HPLC fractions of urine from two coal smoke-exposed women at highrisk for lung cancer demonstrated the presence of 20 and 50 fmol BP-6-N7Gua per mL of urine.Unexposed controls were negative. With proposed modifications, the IA-based protocol canachieve a detection limit of 0.1 fmol/mL urine, which is sufficient for routine quantification ofBP-adducted bases in urine of cigarette smokers. This procedure may allow screening of personsat risk for lung cancer associated with exposure to PAH in cigarette and other forms of smoke.