title: Determination of DNA adducts by combining acid-catalyzed hydrolysis and chromatographic analysis of the carcinogen-modified nucleobases.
abstract: The commonly used method of analyzing carcinogen-induced DNA adducts involves the hydrolysis of carcinogen-modified DNA samples by using a mixture of enzymes, followed by (32)P-postlabeling or liquid chromatography (LC)-based analyses of carcinogen-modified mononucleotides/nucleosides. In the present study, we report the development and application of a new approach to DNA adduct analysis by combining the H(+)/heat-catalyzed release of carcinogen-modified nucleobases and the use of LC-based methods to analyze DNA adducts. Results showed that heating the carcinogen-modified DNA samples at 70 °C for an extended period of 4 to 6 h in the presence of 0.05% HCl can efficiently induce DNA depurination, releasing the intact carcinogen-modified nucleobases for LC analyses. After optimizing the hydrolysis conditions, DNA samples with C8- and N (2) -modified 2'-deoxyguanosine, as well as N (6) -modified 2'-deoxyadenosine, were synthesized by reacting DNA with 1-nitropyrene, acetaldehyde, and aristolochic acids, respectively. These samples were then hydrolyzed, and the released nucleobase adducts were analyzed using LC-based analytical methods. Analysis results demonstrated a dose-dependent release of target DNA adducts from carcinogen-modified DNA samples, indicating that the developed H(+)/heat-catalyzed hydrolysis method was quantitative. Comparative studies with enzymatic digestion method on carcinogen-modified DNA samples revealed that the two hydrolysis methods did not yield systematically different results.
authors.affiliation: Department of Chemistry, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong SAR, China.
authors.forename: Elvis M K
authors.initials: EM
authors.lastname: Leung
authors.affiliation: Environmental Science Programs, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong SAR, China.
authors.forename: Kailin
authors.initials: K
authors.lastname: Deng
authors.affiliation: Department of Chemistry, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong SAR, China.
authors.forename: Tin-Yan
authors.initials: TY
authors.lastname: Wong
authors.affiliation: Department of Chemistry, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong SAR, China. chanwan@ust.hk.|Environmental Science Programs, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong SAR, China. chanwan@ust.hk.
authors.forename: Wan
authors.initials: W
authors.lastname: Chan
mesh_terms: D000143:Acids / Q000737:chemistry; D002273:Carcinogens / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D018736:DNA Adducts / Q000737:chemistry*; D006868:Hydrolysis
chemical_list: D000143:Acids; D002273:Carcinogens; D018736:DNA Adducts
keywords: 1-Nitropyrene; Acetaldehyde; Aristolochic acids; DNA adduct; DNA hydrolysis; LC-MS analysis