Arif Jamal M, Dresler Carolyn, Clapper Margie L, Gairola C Gary, Srinivasan Cidambi, Lubet Ronald A, Gupta Ramesh C
Biological and Medical Research, King Faisal Specialist Hospital and Research Center, Riyadh 11211, Saudi Arabia.
Chem Res Toxicol. 2006 Feb;19(2):295-9. doi: 10.1021/tx0502443.
Several studies have reported the presence of DNA adducts derived from benzo(a)pyrene and other polyaromatics by 32P-postlabeling/TLC by measuring diagonal radioactive zones (DRZs) in lung tissues of human smokers. However, our experimental studies in rodent models, which used modified chromatographic conditions to obtain distinct adduct spots, suggested that cigarette smoke-related lipophilic DNA adducts may not be derived from polycyclic aromatic hydrocarbons (PAHs) or aromatic amines. In the present study, we have performed similar analysis of human lung tissues to study the chemical nature of DNA adducts. Fifty human lung tissues from cancer patients (ages 42-83 years) with active, ex-, or never-smoking status were analyzed for highly lipophilic DNA adducts by nuclease P1- and n-butanol enrichment-mediated 32P-postlabeling assay. All DNA samples yielded low to highly intense adduct DRZs when adducts were resolved by PEI-cellulose TLC in standard high-salt, high-urea solvents. Adduct burden ranged from 6.6 to 2930 per 10(10) nucleotides. However, when adducts were resolved in a different solvent system comprising of high-salt, high-urea in direction 3 and dilute ammonium hydroxide in direction 4, which retained adducts derived from PAHs and aromatic amines on the chromatograms, this yielded no detectable adducts from human lung DNAs. Furthermore, analysis of human lung DNAs mixed with reference adducted DNAs in multisolvent systems confirmed an absence of PAH- and aromatic amine-derived adducts in human smoker lung DNA. To determine the origin of cigarette smoke-associated DNA adducts, calf thymus DNA was incubated with formaldehyde and acetaldehyde, which are known to be present in cigarette smoke in significant quantities. Analysis of purified DNAs by 32P-postlabeling resulted in adduct DRZs in the aldehyde-modified DNAs when adducts were resolved in standard urea-containing solvents, but no adducts were detected when the ammonium hydroxide-based solvent was used, suggesting that even nonpolyaromatic electrophiles can result in adduct DRZs on the chromatograms similar to those from PAH metabolites. Taken together, our data demonstrate that cigarette smoke-associated lung DNA adducts appear on chromatograms as DRZs, consistent with the literature, but they are not related to PAHs and aromatic amines.
多项研究报告称，通过32P后标记/TLC法，通过测量人类吸烟者肺组织中的对角放射性区域（DRZs），发现了源自苯并(a)芘和其他多环芳烃的DNA加合物。然而，我们在啮齿动物模型中的实验研究使用了改良的色谱条件以获得不同的加合物斑点，结果表明与香烟烟雾相关的亲脂性DNA加合物可能并非源自多环芳烃（PAHs）或芳香胺。在本研究中，我们对人类肺组织进行了类似分析，以研究DNA加合物的化学性质。通过核酸酶P1和正丁醇富集介导的32P后标记分析法，对50例来自癌症患者（年龄42 - 83岁）、有当前吸烟、既往吸烟或从不吸烟状态的人类肺组织进行了高度亲脂性DNA加合物分析。当在标准高盐、高尿素溶剂中通过PEI - 纤维素TLC分离加合物时，所有DNA样品产生的加合物DRZs强度从低到高。加合物负荷范围为每10(10)个核苷酸6.6至2930个。然而，当在由第3方向的高盐、高尿素和第4方向的稀氢氧化铵组成的不同溶剂系统中分离加合物时，该系统在色谱图上保留了源自PAHs和芳香胺的加合物，但在人类肺DNA中未检测到可检测的加合物。此外，在多溶剂系统中对与参考加合DNA混合的人类肺DNA进行分析，证实人类吸烟者肺DNA中不存在源自PAH和芳香胺的加合物。为了确定与香烟烟雾相关的DNA加合物的来源，将小牛胸腺DNA与甲醛和乙醛一起孵育，已知这些物质在香烟烟雾中大量存在。通过32P后标记对纯化的DNA进行分析，当在含尿素的标准溶剂中分离加合物时，醛修饰的DNA中产生了加合物DRZs，但使用基于氢氧化铵的溶剂时未检测到加合物，这表明即使是非多环芳烃亲电试剂也可在色谱图上产生与PAH代谢产物类似的加合物DRZs。综上所述，我们的数据表明，与香烟烟雾相关的肺DNA加合物在色谱图上表现为DRZs，这与文献一致，但它们与PAHs和芳香胺无关。
