Shields P G, Kato S, Bowman E D, Petruzzelli S, Cooper D P, Povey A C, Weston A
Laboratory of Human Carcinogenesis, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
IARC Sci Publ. 1993(124):243-54.
Methods for the detection of carcinogen-DNA adducts require both sensitivity and specificity. The 32P-postlabelling assay is highly sensitive but lacks adduct specificity. The strategy reported herein combines micropreparative techniques including HPLC and immunoaffinity chromatography (IAC) to enhance chemical specificity. The resultant assays have retained sensitivity for human DNA analysis. Combined HPLC and 32P-postlabelling has detected 7-methyldeoxyguanosine in human lung samples, while combined IAC and 32P-postlabelling has detected O6-methyldeoxyguanosine adducts in stomach tissue. The limits of detection are one adduct in 10(7) and 10(8) unmodified deoxyguanosine (dG), respectively. IAC was combined with 32P-postlabelling to detect polycyclic aromatic hydrocarbon (PAH)-DNA adducts in human lung. The detection limit was one adduct in 10(7) dG. Our laboratory has also employed synchronous fluorescence spectroscopy (SFS) for the detection of adducts formed from benzo[a]pyrene in human lung. Complex fluorescence matrices suggest the presence of other PAH-DNA adducts. Both the SFS assay and the 32P-postlabelling assay were applied to a series of human samples and a high correlation was found for adduct levels. The development of such assays using synthetic standards, internal standards, determination of calibration curves and validation with corroborating methods is required for chemically specific and sensitive adduct detection.
检测致癌物 - DNA加合物的方法既需要灵敏度又需要特异性。32P后标记分析法灵敏度很高，但缺乏加合物特异性。本文报道的策略结合了包括高效液相色谱（HPLC）和免疫亲和色谱（IAC）在内的微量制备技术，以提高化学特异性。所得分析方法保留了对人类DNA分析的灵敏度。HPLC与32P后标记相结合已在人肺样本中检测到7 - 甲基脱氧鸟苷，而IAC与32P后标记相结合已在胃组织中检测到O6 - 甲基脱氧鸟苷加合物。检测限分别为每10(7)和10(8)个未修饰的脱氧鸟苷（dG）中有一个加合物。IAC与32P后标记相结合用于检测人肺中的多环芳烃（PAH） - DNA加合物。检测限为每10(7)个dG中有一个加合物。我们实验室还采用同步荧光光谱法（SFS）检测人肺中由苯并[a]芘形成的加合物。复杂的荧光图谱表明存在其他PAH - DNA加合物。SFS分析和32P后标记分析都应用于一系列人类样本，发现加合物水平具有高度相关性。为了进行化学特异性和灵敏的加合物检测，需要使用合成标准品、内标、校准曲线的测定以及用确证方法进行验证来开发此类分析方法。
